Micro-devices for improved disease detection

ABSTRACT

The present invention provides a method for detecting a disease at a very low concentration of diseased biological subject by contacting the diseased biological subject with a micro-device which comprises: a first sorting unit capable of directly detecting an intrinsic property of the biological subject at the microscopic level and sorting the biological subject by the detected intrinsic property; a first detection unit capable of detecting the same or different property of the sorted biological subject at the microscopic level; and a first layer of material having an exterior surface and an interior surface, wherein the interior surface defines a first channel in which the biological subject flows through the first sorting unit, and then the sorted biological subject flows from the first sorting unit to the first detection unit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/789,799, filed on Mar. 8, 2013, which claims priority to U.S.application No. 61/608,363, filed on Mar. 8, 2012, the contents of bothof which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Many serious diseases with high morbidity and mortality, includingcancer and heart diseases, are very difficult to diagnose early andaccurately. Current disease diagnosis technologies typically rely onmacroscopic data and information such as body temperature, bloodpressure, and scanned images of the body. To detect serious diseasessuch as cancer, many of the diagnosis apparatus commonly used today arebased on imaging technologies, including x-ray, CT scan, and nuclearmagnetic resonance (NMR). While they provide various degrees ofusefulness in disease diagnosis, most of them cannot provide accurate,totally safe, and cost-effective diagnosis of such serious diseases ascancer at an early stage. Further, many of the existing diagnosistechniques and related apparatus are invasive and sometimes not readilyaccessible, especially in remote regions or rural areas.

Even the newly emerged technologies such as those deployed in DNA testshave not been proven effective in diagnosing a wide range of diseases ina rapid, reliable, accurate, and cost-effective manner. In recent years,there have been some efforts in using nano technologies for variousbiological applications, with most of the work focused on gene mappingand moderate developments in the field of disease detection. Forinstance, Pantel et al. discussed the use of a MicroEelectroMechanicalSystems (MEMS) sensor for detecting cancer cells in blood and bonemarrow in vitro (see, e.g., Klaus Pantel et al., Nature Reviews, 2008,8, 329); Kubena et al. disclose in U.S. Pat. No. 6,922,118 thedeployment of MEMS for detecting biological agents; and Weissman et al.disclose in U.S. Pat. No. 6,330,885 utilizing MEMS sensor for detectingaccretion of biological matter.

However, to date, most of the above described technologies have beenlimited to isolated examples for sensing, using systems of relativelysimple constructions and large dimensions but often with limitedfunctions, and lack sensitivities and specificities. Further, someexisting technologies utilizing nano-particles and biological approacheshave the drawbacks of requiring complicated sample preparationprocedures (such as using chemical or biological markers), difficulty indata interpretation, and too much reliance on visual and color change asmeans of diagnosis (which is subjective and of limited resolution),making them unsuitable for early stage disease detection, e.g., for suchserious diseases as cancer, and particularly for routine hospitalscreening and/or regular physical check-up examinations. Some cannotachieve high degree of sensitivity and specificity simultaneously.

These drawbacks call for novel solutions that not only overcome them butalso bring improved accuracy, sensitivity, specificity, efficiency,non-invasiveness, practicality, simplicity, and speed in early-stagedisease detection at reduced costs.

SUMMARY OF THE INVENTION

The present invention in general relates to a class of innovative andintegrated micro-devices for carrying out disease detection atmicroscopic levels, in vivo or in vitro, on a single cell, a singlebiological molecular (e.g., DNA, RNA, or protein), a single biologicalsubject (e.g., a single virus), or other sufficiently small unit orfundamental biological composition. This class of micro-devices can bemade by using state-of-the-art micro-device fabrication technologies andnovel process flows such as integrated circuit fabrication technologies.As used herein, the term “disease detection apparatus” can beinterchanged with such terms as disease detection device or apparatusintegrated with micro-devices, or any other similar terms of the samemeaning. The micro-devices of this invention contain multiple microunits to perform different functions and optionally detect multipleparameters of a biological subject to be detected or analyzed. Optionalcomponents of the apparatus includes means to perform at least thefunction of addressing, controlling, forcing, receiving, amplifying,manipulating, processing, analyzing, making decisions (e.g., logicdecisions), or storing information from each probe. Such means can be,e.g., a central control unit that includes a controlling circuitry, anaddressing unit, an amplifier circuitry, a logic processing circuitry,an analog device, a memory unit, an application specific chip, a signaltransmitter, a signal receiver, or a sensor.

These disease detection micro-devices are capable of detecting diseasesat their early stages with a higher and much improved degree ofsensitivity, specificity, speed, simplicity, practicality, convenience(e.g., simpler operating procedures or reduced apparatus size), oraffordability (e.g., reduced costs), with substantially reduced to noinvasiveness and side effects. Accordingly, the micro-devices of thisinvention are capable of perform at a much higher level than those ofconventional disease detection apparatus or technologies.

Examples of inventive fabrication techniques or processes that can beused to make the micro-devices of this invention include, but are notlimited to, mechanical, chemical, physical-chemical, chemicalmechanical, electrical, physical, bio-chemical, bio-physical,bio-physical mechanical, electro-mechanical, bio-electro-mechanical,micro-electro-mechanical, electro-chemical-mechanical,electro-bio-chemical-mechanical, nano-fabrication techniques, integratedcircuit and semiconductor manufacturing techniques and processes. For ageneral description of some of the applicable fabrication technologies,see, e.g., R. Zaouk et al., Introduction to Microfabrication Techniques,in Microfluidic Techniques (S. Minteer, ed.), 2006, Humana Press;Microsystem Engineering of Lab-on-a-chip Devices, 1st Ed. (Geschke,Klank & Telleman, eds.), John Wiley & Sons, 2004. Micro-devicefunctionalities would at least include sensing, detecting, measuring,diagnosing, monitoring, and analyzing for disease diagnosis. Multiplemicro-devices can be integrated onto a piece of detection apparatus tomake the apparatus more advanced and sophisticated for further enhancedmeasurement sensitivity, specificity, speed and functionalities, withability to measure the same parameter or a set of different parameters.

Specifically, one aspect of this invention provides micro-devices fordetecting a disease in a biological subject with improved accuracy,sensitivity, specificity, efficiency, non-invasiveness, practicality,simplicity, or speed, at reduced costs and tool size. Each micro-deviceincludes:

a first sorting unit capable of detecting a property of the biologicalsubject at the microscopic level and sorting the biological subject bythe detected property;

a first detection unit capable of detecting the same or differentproperty of the sorted biological subject at the microscopic level; and

a first layer of material having an exterior surface and an interiorsurface, wherein the interior surface defines a first channel in whichthe biological subject flows from the first sorting unit to the firstdetection unit;

wherein the first sorting unit and the first detection unit areintegrated into the first layer of material and positioned to be atleast partially exposed in the channel.

In some embodiments, the micro-device further includes a second sortingunit, wherein the biological subject flows by the first sorting unitbefore reaching the second sorting unit, and the second sorting unit iscapable of detecting the same or different property of the biologicalsubject as the first sorting unit and further sorting the biologicalsubject by the property it detects. Alternatively, the micro-device mayfurther include a second detection unit, wherein the biological subjectflows by the first detection unit before reaching the second detectionunit, and the second detection unit is capable of detecting the same ordifferent property of the biological subject as the first detectionunit. Optionally, a portion of the biological subject from the exit ofsorting unit, which is a likely suspect of diseased biological subject,continues to flow to the detection unit, while the rest of thebiological subject is directed to another direction for separatedisposal (e.g., being dispelled to another exit as waste or for othertypes of tests).

In some embodiments, the biological subject that flows out of thedetection unit is transported back to the sorting unit for furthersorting and detection of a same or different property at the microscopiclevel. This process can be repeated to further concentrate the number ofsuspected, diseased biological entity (e.g., to increase the number ofthe diseased biological entities to be detected per unit volume).

In some embodiments, each property to be detected by a sorting unit or adetection unit is independently an electrical, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,electro-mechanical, electro-chemical, electro-optical, electro-thermal,electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-optical,bio-thermal, bio-physical, bio-electro-mechanical, bio-electro-chemical,bio-electro-optical, bio-electro-thermal, bio-mechanical-optical,bio-mechanical thermal, bio-thermal-optical,bio-electro-chemical-optical, bio-electro-mechanical-optical,bio-electro-thermal-optical, bio-electro-chemical-mechanical, physicalor mechanical property, or a combination thereof. For example, theelectrical property can be surface charge, surface potential, restingpotential, electrical current, electrical field distribution, electricaldipole, electrical quadruple, three-dimensional electrical or chargecloud distribution, electrical properties at telomere of DNA andchromosome, capacitance, or impedance; the thermal property can betemperature or vibrational frequency; the optical property can beoptical absorption, optical transmission, optical reflection,optical-electrical property, brightness, or fluorescent emission; thechemical property can be pH value, chemical reaction, bio-chemicalreaction, bio-electro-chemical reaction, reaction speed, reactionenergy, speed of reaction, oxygen concentration, oxygen consumptionrate, ionic strength, catalytic behavior, chemical additives to triggerenhanced signal response, bio-chemical additives to trigger enhancedsignal response, biological additives to trigger enhanced signalresponse, chemicals to enhance detection sensitivity, bio-chemicals toenhance detection sensitivity, biological additives to enhance detectionsensitivity, or bonding strength; the physical property can be density,shape, volume, or surface area; the biological property can be surfaceshape, surface area, surface charge, surface biological property,surface chemical property, pH, electrolyte, ionic strength, resistivity,cell concentration, or biological, electrical, physical or chemicalproperty of solution; the acoustic property can be frequency, speed ofacoustic waves, acoustic frequency and intensity spectrum distribution,acoustic intensity, acoustical absorption, or acoustical resonance; themechanical property can be internal pressure, hardness, flow rate,viscosity, shear strength, elongation strength, fracture stress,adhesion, mechanical resonance frequency, elasticity, plasticity, orcompressibility.

In some embodiments, the sorting unit and the detection unit eachcomprise a first sensor positioned to be partially in the channel andcapable of detecting a property of the biological subject at themicroscopic level, wherein the property to be detected by the sensors inthe sorting unit and the detection unit can be the same or different.

In some embodiments, at least one of the sensors, the sorting units andthe detection units is fabricated by microelectronics technologies. Forinstance, the sensors can be fabricated to be integral part of theinterior surface that defines the first channel, or the sensors can befabricated separately from and then bonded to the interior surface thatdefines the first channel.

In some embodiments, at least one of the sensors is positioned throughthe exterior and interior surfaces of the first layer of material andexposed in the channel defined by the interior surface and the spaceoutside the exterior surface.

In some embodiments, the first sensor is connected to a circuitryoutside the exterior surface.

In some embodiments, the sorting unit or the detection unit furthercomprises a read-out circuitry which is connected to the first sensorand transfers data from the first sensor to a recording device. Theconnection between the read-out circuit and the first sensor can bedigital, analog, optical, thermal, piezo-electrical, piezo-photronic,piezo-electrical photronic, opto-electrical, electro-thermal,opto-thermal, electrical, electromagnetic, electromechanical, ormechanical.

In some embodiments, the sorting unit or the detection unit furthercomprises at least one additional sensor which is positioned in theinterior surface defining the channel and capable of detecting the sameor different property as the first sensor. For example, the sorting unitor the detection unit may further comprise at least three additionalsensors each of which is positioned in the same interior surfacedefining the channel and detects the same or different property as thefirst sensor. These sensors can be arranged in one group or at least twogroups.

In some embodiments, each sensor is independently an electrical sensor,magnetic sensor, electromagnetic sensor, thermal sensor, optical sensor,acoustical sensor, biological sensor, chemical sensor,electro-mechanical sensor, electro-chemical sensor, electro-opticalsensor, electro-thermal sensor, electro-chemical-mechanical sensor,bio-chemical sensor, bio-mechanical sensor, bio-optical sensor,bio-thermal sensor, bio-physical sensor, bio-electro-mechanical sensor,bio-electro-chemical sensor, bio-electro-optical sensor,bio-electro-thermal sensor, bio-mechanical-optical sensor,bio-mechanical thermal sensor, bio-thermal-optical sensor,bio-electro-chemical-optical sensor, bio-electro-mechanical opticalsensor, bio-electro-thermal-optical sensor,bio-electro-chemical-mechanical sensor, physical sensor, mechanicalsensor, piezo-electrical sensor, piezo-electro photronic sensor,piezo-photronic sensor, piezo-electro optical sensor, bio-electricalsensor, bio-marker sensor, image sensor, or radiation sensor. Forexample, the thermal sensor can comprise a resistive temperaturemicro-sensor, a micro-thermocouple, a thermo-diode andthermo-transistor, and a surface acoustic wave (SAW) temperature sensor;the image sensor comprises a charge coupled device (CCD) or a CMOS imagesensor (CIS); the radiation sensor can comprise a photoconductivedevice, a photovoltaic device, a pyro-electrical device, or amicro-antenna; the mechanical sensor can comprise a pressuremicro-sensor, micro-accelerometer, flow meter, viscosity measurementtool, micro-gyrometer, or micro flow-sensor; the magnetic sensor cancomprise a magneto-galvanic micro-sensor, a magneto-resistive sensor, amagneto diode, or magneto-transistor; the biochemical sensor cancomprise a conductimetric device or a potentiometric device.

In some embodiments, at least one sensor is a probing sensor and canapply a probing or disturbing signal to the biological subject to betested. Optionally, at least one sensor (i.e., not the just-mentionedprobing sensor) or another sensor (along with the just-mentioned probingsensor) is a detection sensor and detects a response from the biologicalsubject upon which the probing or disturbing signal is applied.

In some embodiments, the one or more sensors are fabricated on theinterior surface of the layer of material. For example, at least twosensors can be fabricated on the interior surface of the layer ofmaterial and are arranged in an array.

In some embodiments, the channel defined by the interior surface has asymmetric configuration, e.g., an oval, circular, triangular, square, orrectangular configuration. In some particular embodiments, the channelhas a rectangular configuration and 4 sides of walls.

In some embodiments, the channel has a length ranging from 1 micron to50,000 microns.

In some embodiments, at least two sensors are located on one side or twoopposite sides of the interior surface defining the channel. Forexample, at least four sensors can be located on one side, two oppositesides, or four sides of the interior surface defining the channel.

In some embodiments, the sorting unit or the detection unit comprise twopanels, at least one of the two panels is fabricated by microelectronictechnologies and comprises a read-out circuitry and a sense, and thesensor is positioned on the interior surface which defines the channel.

In some embodiments, the sorting unit or the detection unit furthercomprises two micro-cylinders that are placed between and bonded withthe two panels, wherein each of the micro-cylinders is solid, hollow, orporous, and optionally fabricated by microelectronics technologies.

In some embodiments, the micro-cylinders are solid and at least one ofthem comprises a sensor fabricated by microelectronics technologies. Thesensor in the micro-cylinder can detect the same or different propertyas a sensor in the panel does.

In some embodiments, the sensor in the micro-cylinder applies a probingsignal to the biological subject.

In some embodiments, at least one of the micro-cylinders comprises atleast two sensors fabricated by microelectronics technologies, and everytwo of the at least two sensors are so located in the micro-cylinder toform an array of the sensors on the panel.

In some embodiments, the two sensors in the micro-cylinder are apart bya distance ranging from 0.1 micron to 500 microns, from 0.1 micron to 50microns, form 1 micron to 100 microns, from 2.5 microns to 100 microns,or from 5 microns to 250 microns.

In some embodiments, at least one of the panels comprises at least twosensors that are arranged in at least two arrays each separated by atleast a micro sensor in a cylinder.

In some embodiments, at least one array of the sensors in the panelcomprises two or more sensors.

In some embodiments, the sorting unit or the detection unit furthercomprises an application specific integrated circuit chip which isinternally bonded to or integrated into one of the panels or amicro-cylinder.

In some embodiments, the sorting unit or the detection unit furthercomprises an optical device, imaging device, camera, viewing station,acoustic detector, piezo-electrical detector, piezo-photronic detector,piezo-electro photronic detector, electro-optical detector,electro-thermal detector, electrical detector, bio-electrical detector,bio-marker detector, bio-chemical detector, chemical sensor, thermaldetector, ion emission detector, or thermal recorder, each of which isintegrated into the a panel or a micro cylinder.

In some embodiments, the interior surface defines at least oneadditional channel for transporting and sorting or detecting thebiological subject.

In some embodiments, the interior surface defines at least oneadditional channel for transporting away a portion of the biologicalsubject that is an unlikely suspect of being diseased based on thesorting and/or detection.

In some embodiments, the interior surface defines at least oneadditional channel for transporting the biological subjects suspected ofdisease based on the sorting and/or detection for further sorting and/ordetection. The further sorting and/or detection may include, e.g.,transporting such a suspected biological subject back to the sortingunit and/or detection unit where it has been processed for furtherconcentration (i.e., increasing the number of diseased biologicalsubject, or the number of diseased biological entities in the biologicalsubject, per unit volume).

In some embodiments, the micro-device has numerous (e.g., from a few tohundreds or millions) channels for transporting and sorting or detectingthe biological subject.

In some embodiments, the channel has a diameter or height or widthranging from 0.1 micron to 150 microns, from 0.5 micron to 5 microns,from 1 micron to 2.5 microns, from 3 microns to 15 microns, from 5microns to 25 microns, from 5 microns to 50 microns, from 25 microns to50 microns, or from 50 microns to 80 microns; and the channel has alength ranging from 0.5 micron to 50,000 microns.

In some embodiments, the sorting unit or the detection unit comprisesand is capable of releasing a bio-marker, a nano-particle, a magneticparticle, or a nano-particle attached to a bio-marker, or a combinationthereof, for mixing with and sorting or detecting the biologicalsubject.

In some embodiments, the nano-particle attached to a bio-marker is amagnetic nano-particle; and one or more magnetic nano-particles aremixed with the biological subject for separating and detecting thebiological subject. For example, the bio-marker can be attached with alight emitting item and mixed with the biological subject. The lightemitting item can be a florescence generating component.

In some embodiments, the mixed biological subject flows through achannel; a signal of the mixed biological subject is detected andcollected by a sensor in a sorting or detection unit; and the signal isan electrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-optical, electro-thermal, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-optical, bio-thermal, bio-physical,bio-electro-mechanical, bio-electro-chemical, bio-electro-optical,bio-electro-thermal, bio-mechanical-optical, bio-mechanical thermal,bio-thermal-optical, bio-electro-chemical-optical,bio-electro-mechanical-optical, bio-electro-thermal-optical,bio-electro-chemical-mechanical, physical or mechanical signal, or acombination thereof.

In some embodiments, the biological subject flows through the firstchannel and, after the sorting unit, is separated into a suspectedcomponent and an unsuspected component, and the two components continueto flow through the channel in two different directions.

In some embodiments, the micro-device of this invention furthercomprises one or more additional channels each of which is defined bythe interior surface of the first or additional layer of material and isintegrated to the first channel, and the separated suspected componentor unsuspected component flows through the additional channel(s) forfurther separation.

In some embodiments, the micro-device of this invention furthercomprises multiple additional channels, each of the additional channelsis defined by the interior surface of the first layer of material oradditional layer(s) of material, is directly or indirectly integrated tothe first channel and other channel(s), and optionally comprises asorting unit or a detection unit attached to the interior surfacedefining the channel; and the biological subject flows through thesemultiple channels simultaneously and are sorted and separated therein.

In some embodiments, the first channel is centrally positioned in themicro-device as compared to the other additional channels and isconnected to at least two other channels; and a designed componentinjected into the first channel flows from this first channel to theother connected channels.

In some embodiments, the designed component is a bio-marker, anano-particle, a magnetic particle, or a nano-particle attached to abio-marker, a disturbing fluid, or a combination thereof.

In some embodiments, the amount, timing or speed of the designedcomponent injected into the first channel is pre-programmed orcontrolled in real time.

In some embodiments, the micro-device of this invention furthercomprises a probing unit which is capable of applying a probing ordisturbing signal to the biological subject or a media in which thebiological subject is contained, thereby changing the nature or value ofa property of the biological subject or of the media.

In some embodiments, the probing signal can be of the same or differenttype as the property to be detected and can change the value of theproperty to be detected. The probing signal or the property to bedetected can be independently an electrical, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-optical, electro-thermal,electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-optical,bio-thermal, bio-physical, bio-electro-mechanical, bio-electro-chemical,bio-electro-optical, bio-electro-thermal, bio-mechanical-optical,bio-mechanical thermal, bio-thermal-optical,bio-electro-chemical-optical, bio-electro-mechanical-optical,bio-electro-thermal-optical, bio-electro-chemical-mechanical, physicalor mechanical property, or a combination thereof. The electricalproperty can be surface charge, surface potential, resting potential,electrical current, electrical field distribution, electrical dipole,electrical quadruple, three-dimensional electrical or charge clouddistribution, electrical properties at telomere of DNA and chromosome,capacitance, or impedance; the thermal property can be temperature orvibrational frequency; the optical property can be optical absorption,optical transmission, optical reflection, optical-electrical property,brightness, or fluorescent emission; the chemical property can be pHvalue, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, speed of reaction, oxygenconcentration, oxygen consumption rate, ionic strength, catalyticbehavior, chemical additives to trigger enhanced signal response,bio-chemical additives to trigger enhanced signal response, biologicaladditives to trigger enhanced signal response, chemicals to enhancedetection sensitivity, bio-chemicals to enhance detection sensitivity,biological additives to enhance detection sensitivity, or bondingstrength; the physical property can be density, shape, volume, orsurface area; the biological property can be surface shape, surfacearea, surface charge, surface biological property, surface chemicalproperty, pH, electrolyte, ionic strength, resistivity, cellconcentration, or biological, electrical, physical or chemical propertyof solution; the acoustic property can be frequency, speed of acousticwaves, acoustic frequency and intensity spectrum distribution, acousticintensity, acoustical absorption, or acoustical resonance; themechanical property can be internal pressure, hardness, flow rate,viscosity, shear strength, elongation strength, fracture stress,adhesion, mechanical resonance frequency, elasticity, plasticity, orcompressibility.

In some embodiments, the probing signal is changed from a static valueto a dynamic value or to a pulsed value, or from a lower value to ahigher value.

In some embodiments, at least one of the properties of the media ischanged from a static value to a dynamic value or to a pulsed value, orfrom a lower value to a higher value.

In some embodiments, the probing signal or a property of the media islaser intensity, temperature, catalyst concentration, acoustic energy,bio-maker concentration, electrical voltage, electrical current,fluorescent dye concentration, the amount of agitation of the biologicalsample, or fluid flow rate.

In some embodiments, the micro-device of this invention furthercomprises a pre-screening unit which is capable of pre-screening adiseased biological subject from a non-diseased biological subject basedon the difference in a property between a diseased biological subjectand a non-diseased biological subject.

In some embodiments, the disease to be detected is a cancer. Examples ofthe cancer include breast cancer, lung cancer, esophageal cancer,intestine cancer, cancer related to blood (e.g., leukemia), livercancer, and stomach cancer. Yet, additional examples include circulatingtumor cells (CTCs) which are very important and can occur in late stagecancer patients (sometime, they occur after cancer treatment relatedsurgeries).

Another aspect of this invention provides methods for detecting adisease at a very low concentration of diseased biological subject. Eachmethod comprises contacting the diseased biological subject with amicro-device which comprises:

a first sorting unit capable of detecting a property of the biologicalsubject at the microscopic level and sorting the biological subject bythe detected property;

a first detection unit capable of detecting the same or differentproperty of the sorted biological subject at the microscopic level; and

a first layer of material having an exterior surface and an interiorsurface, wherein the interior surface defines a first channel in whichthe biological subject flows from the first sorting unit to the firstdetection unit;

wherein the first sorting unit and the first detection unit areintegrated into the first layer of material and positioned to be atleast partially exposed in the channel.

In some embodiments of the methods, the diseased biological subject iscells, a sample of an organ or tissue, DNA, RNA, virus, or protein.

In some embodiments of the methods, the cells are circulating tumor orcancer cells.

In some embodiments of the methods, the biological subject is containedin a media and transported into the first channel of micro-device.

In some embodiments of the methods, the micro-device further comprises aprobing unit which is capable of applying a probing signal to thebiological subject or a media in which the biological subject iscontained, thereby changing the nature or value of a property of thebiological subject or of the media.

In some embodiments of the methods, the micro-device further comprisinga pre-screening unit which is capable of pre-screening a diseasedbiological subject from a non-diseased biological subject based on thedifference in a property between a diseased biological subject and anon-diseased biological subject.

Still another aspect of this invention provides apparatus for detectinga disease, each comprising a first micro-device and a first substratesupporting the first micro-device, wherein the first micro-devicecontacts a biological subject to be analyzed and is capable of measuringat the microscopic level an electrical, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-physical, bio-mechanical, bio-thermal, bio-optical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, photo-electrical, physical, ormechanical property, or a combination thereof, of the biologic material.The apparatus can further optionally include a device for reading thedata from measuring the property. The difference in the measuredproperty of the tested biologic material and that of a biologic samplefrom a subject free of the disease is indicative of the possibleoccurrence of the disease in early stage.

In some other embodiments, the electrical property is surface charge,surface potential, oscillation in electrical signal (e.g., oscillationin ions, pulsing electrical field, pulsing surface charge, pulsingvoltage), capacitance, electro-magnetic parameters, electrical field,electrical field distribution, electrical charge distribution, orimpedance; the thermal property is temperature; the chemical property ispH value, ionic strength, bonding strength; the physical property isdensity, flow rate, volume, and surface area; and the mechanicalproperty is hardness, shear strength, elongation strength, fracturestress, adhesion, elasticity, or density. These properties can be staticor dynamic. For example, an electrical current can be a constant current(DC) or an alternating current (AC). They can also be measured andrecorded in their values in a transition period from a static state to adynamic state.

In some embodiments, the probing and detecting device applies to thebiological subject a voltage ranging from about 1 mV to about 10 V, orfrom about 1 mV to about 1.0 V.

In some embodiments, the first micro-device comprises a conductivematerial, an electrically insulating material, a biological material, ora semiconductor material.

In some other embodiments, each of the apparatus further comprises atleast one or more additional micro-devices. In these embodiments, eachof the micro-devices contained in the apparatus comprises a conductivematerial, an electrically insulating material, or a semiconductormaterial; and each of the micro-devices can comprise the same ordifferent material(s) and can measure the same or different propertiesat the same or different time.

In some embodiments, the probing device and the micro-devices are placedwith a desired distance between each other. These multiple micro-devicescan be spaced out, e.g., with a distance of at least 10 angstroms on thesubstrate, or with a distance ranging from about 5 microns to about 100microns.

The multiple micro-devices integrated in a disease detection apparatuscan sequentially and/or simultaneously measure various parameters from abiological subject being detected at macroscopic and/or microscopiclevels. Sometimes, in an apparatus with multiple micro-devices, somemicro-devices can act as probing devices to disturb the biologicalsubject and trigger a response from the biological subject, while othermicro-devices in the apparatus can act as detection devices to measurethe triggered response by the biological subject. Another inventiveaspect of this application is that during measurements, sometimes, atleast one of the parameters applied to the biological sample beingmeasured or at least one of the properties in the surrounding media (inwhich the biological sample resides) is intentionally changed from astatic state (constant value) to a dynamic state (for example, a pulsedvalue or an alternating value), or from one value to a new value. As anexample, in a measurement, a DC current applied to a biological sampleis intentionally changed to an AC current. In another example, aconstant temperature applied to a biological sample is changed to ahigher temperature, or a pulsed heat wave (for example, from 30° C. to50° C., then from 50° C. back to 30° C.).

In some other embodiments, each of the micro-devices has the sizeranging from about 1 angstrom (Å) to about 5 millimeter (e.g., from 5 Åto 1 millimeter).

In some other embodiments, the apparatus comprises one or moreadditional substrates on which the micro-devices are placed. Each of thesubstrates can comprise the same or a different material (e.g., aconductive material or an insulator) and can be in the same or adifferent shape (e.g., a slab or a tube), and each substrate can be atwo- or three-dimensional object. They can take the form of cylinder,rectangle, cube, slabs, cavities, long channels, long and narrowchannels, chambers, chambers with channels connected to it, or any otherdesired shapes and configurations, in order to further improve theirmeasurement sensitivity, specificity, speed, sample size, and reducecost and size.

In terms of detection apparatus to integrate micro-devices, in one noveldetection apparatus design, to increase measurement sensitivity,micro-devices mounted on two slabs separated by a small spacing withsample to be measured between the two said slabs can be used to detectdisease with improved speed, with micro-devices measuring cells, DNAs,and desired items in the sample in parallel. The surface area of theslabs can be maximized in order to have maximum number of micro-devicesplaced on the slabs and enhance measurement efficiency and speed.Optionally, multiple micro-devices integrated on the surface of theslabs can be closely spaced with their spacing matching that of cells,DNAs, and items to be measured.

In another novel configuration, a detection apparatus integrated withmicro-devices is shaped in the form of a cylinder, with multiplemicro-devices with detection probes integrated/mounted in the intersurfaces of the cylinder and with sample to be measured (such as blood,urine, sweat, tear, or saliva) flowing through the cylinder.

In yet another innovative configuration, a detection apparatus withintegrated micro-devices is shaped in the form of a rectangular pipe,with multiple micro-devices with detection probes integrated/mounted inthe inter surfaces of the pipe and with sample to be measured (such asblood, urine, sweat, tear, or saliva) flowing through the rectangularpipe.

In another aspect, the invention provides another set of apparatus fordetecting a disease in a biological subject, comprising a system fordelivering the biological subject to be detected and a probing anddetecting device for probing and detecting the biological subject.

The difference in the measured property of the detected biologicmaterial and of a standard biologic sample is indicative of the possibleoccurrence of the disease.

In some embodiments, the probing and detecting device comprises a firstmicro-device and a first substrate supporting the first micro-device,the first micro-device contacts the biologic subject to be detected andis capable of measuring at the microscopic level an electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-physical, bio-thermal,bio-optical, bio-chemical-physical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, photo-electrical, physical, ormechanical property, or a combination thereof, of the biologic subject.For example, the electrical property can be surface charge, surfacepotential, resting potential, electrical current, electrical fielddistribution, electrical dipole, electrical quadruple, three-dimensionalelectrical or charge cloud distribution, electrical properties attelomere of DNA and chromosome, or impedance; the thermal property canbe temperature, or vibrational frequency of biological item ormolecules; the optical property can be optical absorption, opticaltransmission, optical reflection, optical-electrical property,brightness, or fluorescent emission; the chemical property can be pHvalue, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, or bondingstrength; the physical property can be density or geometric size; theacoustic property is frequency, speed of acoustic waves, acousticfrequency and intensity spectrum distribution, acoustic intensity,acoustical absorption, or acoustical resonance; and the mechanicalproperty is internal pressure, hardness, shear strength, elongationstrength, fracture stress, adhesion, mechanical resonance frequency,elasticity, plasticity, or compressibility.

In some embodiments of the apparatus, the probing and detecting deviceapplies to the biological subject a voltage ranging from about 1 mV toabout 10 V, or from about 1 mV to about 1.0 V.

In some embodiments of the apparatus, the first micro-device comprises aconductive material, an electrically insulating material, a biologicalmaterial, or a semiconductor material.

In some embodiments of the apparatus, the first micro-device has a sizeranging from about 1 angstrom to about 5 millimeter.

In some embodiments of the apparatus, the probing and detecting devicefurther comprises one or more additional micro-devices, each of which isalso capable of measuring at the microscopic level an electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-physical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, photo-electrical, physical, ormechanical property, or a combination thereof, of the biologic entity.

In some embodiments of the apparatus, each of the additionalmicro-devices comprises a conductive material, an electricallyinsulating material, a biological material, or a semiconductor material.Further, each of the additional micro-devices comprises a material thatis the same as or different from the material of the first micro-deviceand is capable of measuring the same or different property of thebiologic subject as the first-micro-device does.

In some embodiments of the apparatus, the first micro-device and each ofthe additional micro-devices can measure the same or differentproperties at the same or different times.

In some embodiments of the apparatus, the probing device and themicro-devices are placed with a desired distance between each other.

In some embodiments of the apparatus, each of the additionalmicro-devices has a size ranging from about 1 angstrom to about 5millimeter.

In some embodiments of the apparatus, the micro-devices are spaced outon the substrate by a distance of at least 10 angstroms (e.g., fromabout 5 microns to about 100 microns).

In some embodiments of the apparatus, the substrate is in the shape of aslab, a rectangle, a cube, a tube, an array of tubes, cavities, longchannels, long and narrow channels, chambers, or chambers with channelsconnected to it; or the substrate is a three-dimensional object.

In some embodiments of the apparatus, the probing and detecting devicefurther comprises a second substrate of the same or different materialas the first substrate.

In some embodiments, the apparatus further comprises a device forreading the data from measuring the property by the probing anddetecting device.

In some embodiments, the apparatus each further comprises a system fordelivering a fluid, which comprises a pressure generator, a pressureregulator, a throttle valve, a pressure gauge, and distributing kits.The pressure generator can include a motor piston system and a bincontaining compressed gas; the pressure regulator can down-regulate orup-regulate the pressure to a desired value; the pressure gauge feedsback the measured value to the throttle valve, which then regulates thepressure to approach the target value.

The fluid to be delivered in the apparatus can be a liquid or gas.Examples of the liquid include blood, urine, saliva, tear, saline, andsweat; whereas examples of the gas include nitrogen, argon, helium,neon, krypton, xenon, or radon.

In some embodiments of the apparatus, the probing and detecting devicefurther comprises a system controller which comprises a pre-amplifier, alock-in amplifier, an electrical meter, a thermal meter, a switchingmatrix, a system bus, a nonvolatile storage device, a random accessmemory, a processor, or a user interface. The interface may include asensor which can be, e.g., a thermal sensor, an electrical sensor, aflow meter, an optical sensor, or a sensor comprising one or morepiezo-electrical materials.

In some embodiments, the apparatus may further include a biologicalinterface, an interface between a sample injector and sample treatmentand/or detection unit, a system controller, or at least one system forreclaiming or treatment medical waste. Reclaiming and treatment ofmedical waste is performed by the same system or by two differentsystems.

In some embodiments, the apparatus further include a testing sampledelivery system, a testing sample distribution system, a distributionchannel, a pre-processing unit, a detection device, a global positioningsystem, a motion device, a signal transmitter, a signal receiver, asensor, a memory storage unit, a logic processing unit, an applicationspecific chip, a testing sample recycling and reclaiming unit, amicro-electro-mechanical device, a multi-functional device, or amicro-instrument to perform surgery, cleaning, or medical function. Suchadditional components each may be fabricated by methods known in theart, e.g., as described in PCT/US2010/041001, PCT/US2011/024672, U.S.application Ser. No. 12/416,280, and PCT/US2011/042637, all of which areincorporated herein by reference in their entireties.

In some embodiments of the apparatus, the system for delivering thebiological subject comprises at least one channel inside which thebiological subject to be detected travels in a certain direction; theprobing and detecting device comprises at least one probing micro-deviceand at least one detecting micro-device, at least one probingmicro-device is located before at least one detecting micro-devicerelative to the direction in which the biological subject travels, andthe probing micro-device and the detecting micro-device can be attachedto the interior or exterior wall of the channel.

In some embodiments, the probing and detecting device comprise at leasttwo detecting micro-devices capable of measuring at the micro-level thesame or different properties of the biological subject.

In some further embodiments, the detecting micro-devices are capable ofmeasuring at the microscopic level the surface charge, surfacepotential, resting potential, action potential, electrical voltage,electrical current, electrical field distribution, electrical chargedistribution, electrical dipole, electrical quadruple, three-dimensionalelectrical or charge cloud distribution, electrical properties attelomere of DNA and chromosome, dynamic changes in electricalproperties, dynamic changes in potential, dynamic changes in surfacecharge, dynamic changes in current, dynamic changes in electrical field,dynamic changes in electrical voltage, dynamic changes in electricaldistribution, dynamic changes in electronic cloud distribution,capacitance, impedance, temperature, vibrational frequency, opticalabsorption, optical transmission, optical reflection, optical-electricalproperty, brightness, fluorescent emission, photo-electrical parameters,pH value, chemical reaction, bio-chemical reaction, bio-electro-chemicalreaction, reaction speed, reaction energy, speed of reaction, oxygenconcentration, oxygen consumption rate, ionic strength, catalyticbehavior, bonding strength, density, geometric size, frequency, speed ofacoustic waves, acoustic frequency and intensity spectrum distribution,acoustic intensity, acoustical absorption, acoustical resonance,internal pressure, hardness, volume, surface area, shear strength,elongation strength, fracture stress, adhesion, mechanical resonancefrequency, elasticity, plasticity, or compressibility. (Weisun, thislist does not seem to be complete—we have a more complete list)

In some embodiments of the apparatus, the shapes and sizes of differentsections of the channel can be the same or different; the width of thechannel ranges from about 1 nm to about 1 mm; the channel can bestraight, curved, or angled; the interior wall of the channel defines acircular, oval, or polygon space; the interior wall of the channeldefines a circular or rectangular space; the channel is a circularcarbon nano-tube.

In some embodiments of the apparatus, the carbon nano-tube has adiameter ranging from about 0.5 nm to about 100 nm and a length rangingfrom about 5.0 nm to about 10 mm.

In some embodiments of the apparatus, the interior wall of the channelhas at least one concave that may be in the same section as a probing ordetecting micro-device. The concave groove can be a cubic space or anangled space; the concave groove can have a depth ranging from about 10nm to about 1 mm.

In some embodiments of the apparatus, a distribution fluid is injectedinto the channel, either before or after the biological subject passes aprobing micro-device, to aid the traveling or separation of thebiological subject inside the channel. The distribution fluid can beinjected into the channel through a distribution fluid channel connectedto an opening in the channel wall.

In some yet other innovative embodiments, a cleaning fluid can be usedto clean the apparatus, particularly narrow and small spaces in theapparatus where biological residues and deposits (such as dried bloodand protein when they are used in or as a sample) likely accumulate andblock such spaces. Desired properties of such a cleaning fluid include,e.g., low viscosity and ability to dissolve the biological residues anddeposits.

The apparatus can be for detecting the diseases of more than onebiological subjects and the channel comprises a device located thereinfor separating or dividing the biological subjects based on differentlevels of a same property of the biological subjects. The separating ordividing device can be, e.g., a slit, and separates or dividesbiological subjects based on their properties such as surface charges.

The apparatus can further include a filtering device for removingirrelevant objects from the biologic subject for detection.

The biological subject can be a DNA, telomere of DNA, RNA, chromosome,cell, cell substructure, protein, tissue, virus, blood, urine, sweat,tear, or saliva.

In some embodiments, the apparatus may further includes a unit fordelivering the biological subject, a channel, a detection unit, a datastorage unit, a data analysis unit, a central control unit, a biologicalsample recirculation unit, a waste disposal unit; a pre-processing unit,a signal processing unit, or a disposal processing unit. All theadditional components can be integrated on a single device or a boardalong with the delivering system and probing and detecting probe. Thepre-processing unit may comprise a sample filtration unit; a deliveryunit for delivering a desired ion, a biological component, or abio-chemical component; a recharging unit; a constant pressure deliveryunit; and a sample pre-probing disturbing unit. The sample filtrationunit may comprise an entrance channel, a disturbing fluid channel, anaccelerating chamber, and a slit. The signal processing unit maycomprise an amplifier, a lock-in amplifier, an A/D (analog-to-digital oralternative to direct electrical current) converter, a micro-computer, amanipulator, a display, and network connections. The signal processingunit may collect more than one signal, collect multiple signalssimultaneously, collect signals simultaneously at different locations,and the signals can be integrated to cancel noise or to enhance thesignal to noise ratio. The collected signal(s) may also be processedthrough one or more lock-in amplifiers to enhance the signal to noiseratio, thereby improving detection sensitivity and repeatability.

In some embodiments of the apparatus, a bio-compatible fluid is injectedinto the disturbing fluid channel to separate the biological subject, orthe bio-compatible fluid is injected from the entrance of the disturbingfluid channel and delivered to an opening in the entrance channel wall.The biocompatible fluid comprises saline, water, an oxygen-rich liquid,or plasma.

In some embodiments of the apparatus, the angle between the entrancechannel and the disturbing fluid channel ranges from about 0° to about180°, from about 30° to about 150°, from about 60° to about 120°, orfrom about 75° to about 105°, or about 90°; the width of each channelranges from about 1 nm to about 1 mm; and at least one of the channelscomprises one probing device attached to the channel's sidewall, whereinthe probing device is capable of measuring at the microscopic level anelectrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject. The sample filtrationunit may comprise an entrance channel, a biocompatible micro-filter, oran exit channel.

In some embodiments of the apparatus, the biocompatible micro-filter iscapable of filtering the biological subject based on at least oneproperty selected from physical size, hardness, elasticity, shearstrength, weight, surface feature, optical, photo-electrical,acoustical, thermal, chemical, physical, mechanical, electrical,biological, bio-chemical, bio-physical, bio-mechanical, bio-electrical,bio-thermal, bio-chemical mechanical, bio-electrical mechanical,bio-optical, bio-electrical optical, bio-chemical optical, electrical,electro-chemical, magnetic, electromagnetic, electro-mechanical,electro-chemical-mechanical, and electro-chemical-biological property.

In some embodiments, at least one of the channels comprises at least twoprobing devices attached to the channel's sidewalls, and the probingdevices are capable of measuring at the microscopic level an electrical,magnetic, electromagnetic, thermal, optical, photo-electrical,acoustical, biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-physical, bio-electrical,bio-mechanical, bio-optical, bio-thermal, physical-chemical,bio-physical, bio-physical mechanical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject.

In some embodiments of the apparatus, the recharging unit rechargesnutrient or respiring gas to the biological subject. The nutrient caninclude a biocompatible strong or weak electrolyte, amino acid, mineral,ions, oxygen, oxygen-rich liquid, intravenous drip, glucose, or protein;and the respiring gas can include oxygen.

In some embodiments, the biological subject to be tested comprisesblood, urine, saliva, tear, saline, or sweat.

In some embodiments, the signal processing unit comprises an amplifier,a lock-in amplifier, an A/D converter, a micro-computer, a manipulator,a display, or a network connection. It can collect more than one signal,and the signals can be integrated to reduce (i.e., cancel out) noise andhence enhance the signal to noise ratio.

In still another aspect, the invention provides alternative apparatusfor detecting a disease in a biological subject. The apparatus eachcomprise a launching chamber to launch a probe object at a desired speedand direction, a detection unit, a probe object, a detection component,a channel for transporting the biological subject to be tested and theprobe object.

In some embodiments of these apparatus, the launching chamber comprisesa piston for releasing the probe object and a channel for directing theprobe object.

In some embodiments, the detection unit or the detection component iscapable of measuring at the microscopic level an electrical, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,physical-chemical, electro-mechanical, electro-chemical,electro-optical, electro-thermal, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-physical-mechanical, bio-optical,bio-thermal, bio-physical, bio-electro-mechanical, bio-electro-chemical,bio-electro-optical, bio-electro-thermal, bio-mechanical-optical,bio-mechanical thermal, bio-thermal-optical,bio-electro-chemical-optical, bio-electro-mechanical-optical,bio-electro-thermal-optical, bio-electro-chemical-mechanical, physicalor mechanical property, or a combination thereof.

Yet still another set of apparatus for detecting a disease in abiological subject as provided by this invention are those fabricated bya method comprising: providing a substrate; sequentially depositing afirst material and a second material as two layers onto the substrate toform a material stack; patterning the second material to form a firstdesired feature; depositing a third material onto the material stack tocover the second material; optionally patterning the first material andthird material to form a second desired feature; and optionallydepositing a fourth material onto the material stack; wherein thedetection device is capable of interacting with the biological subjectto generate a response signal. If desired, in order to enhancefunctionality of the apparatus, density of the components such asdetectors in the apparatus, and measurement speed of the apparatus, oneor more of the above steps can be repeated. In one embodiment, the aboveflow can be repeated to create vertically stacked, multiple layers ofsuch features (components) which allow simultaneous measurements of manybiologic samples to significantly increase the measurement speed andefficiency. This will be useful, e.g., for detecting circulating tumorcells (CTCs) which typically exist at a very low concentration (e.g.,one part per billion).

In some embodiments, in these methods used for fabricating theapparatus, the second material can be patterned by microelectronicprocesses.

In some embodiments, in these methods used for fabricating theapparatus, the first material and third material can be the same ordifferent.

In some embodiments, in these methods used for fabricating theapparatus, the first material and third material are patterned bylithography and etch processes selective to the second material to format least one type of trench feature in the layers of the third materialand first material.

In some embodiments, in these methods used for fabricating theapparatus, the fabrication method may further comprise capping the topof the material stack to form an enclosed trench. The enclosed trenchcan, e.g., be used to observe and record features and behaviors of thebiological subject. The capping can comprise, e.g., placing a seconddevice on the top of the material stack, and the second device can be adevice identical to the detection device being capped, a piece of glassor crystal, or a functional device selected from the group consisting ofan imaging device, a sensor (e.g., an optical sensor), a memory storage,a signal transmission, a logic processing component, a circuit for datastorage, signal transmission, signal receiving, and signal processing.

In some embodiments, in these methods used for fabricating theapparatus, the first feature or second feature is selected from thegroup consisting of partitioned chambers, chambers connected withchannels, channels, probe generator (probe), detection probes,electrically connective interconnection lines, optical transmissionlines, piezo-photonic lines, piezo-electrical photronic lines, andpiezo-electrical lines. For example, the partitioned chambers can be forpre-processing of the biological subject for initial screening andenhancement of concentration of diseased biological subject for furthertesting, chambers connected with channels are for pre-processing anddetection, channels can be for biological subject to flow through, theprobe generator (probe) can be utilized for generating probe and disturbsignal onto the biological subject for triggering a response signal, thedetection probe can be for measuring properties of the biologicalsubject and the response signal, the electrically connectiveinterconnection lines can be for transmitting signals, the opticaltransmission lines can be for transmitting signals, and piezo-electricallines can be for using piezo-electrical effect to probe biologicalsubjects.

In some embodiments, in these methods used for fabricating theapparatus, the second material is patterned using lithography and etchprocesses selective to the first material to form a desired componentsuch as a detection probe.

In some embodiments, in these methods used for fabricating theapparatus, the first and third materials are patterned using lithographyand etch processes selective to the second material to form at least onetype of trench feature in the layers of the third and first materials,with the second material reasonably aligned with the wall of the trench.

In some embodiments, in these methods used for fabricating theapparatus, the thickness of the fourth material is thinner than that ofthe third material.

In some embodiments, the second and the fourth materials form detectionprobes.

In some embodiments, the second and the fourth materials form a probeand a detector, respectively.

In some embodiments, the apparatus may further include an imaging devicefor observing and recording properties and behaviors of the biologicalsubject.

In some embodiments, the apparatus may further include a pre-processingunit with chambers for pre-screening and enhancing a diseased biologicalsubject for further testing, channels for carrying fluidic sample toflow through, probes for probing and disturbing the biological subjectbeing tested for generating response signals, detection probes formeasuring properties and response signals of the biological subject, andan imaging device, a camera, a viewing station, an acoustic detector, athermal detector, an ion emission detector, or a thermal recorder forobserving and recording properties and behaviors of the biologicalsubject.

In some embodiments, the apparatus may further include a memory storageunit, a signal transmission component, a logic processing component, ora circuit for data storage, signal transmission, signal receiving, orsignal processing. These additional devices can be fabricated bymicroelectronics processes on the substrate where the first material isdeposited.

In some embodiments, the apparatus may have typical channel dimensionsranging from about 2 microns×2 microns to about 100 microns×100 micronsin cross sectional area for a square-shaped channel, a rectangle-shapedchannel, a radius ranging from about 1 micron to about 20 microns incross sectional area for a circular shaped channel, and a typical probedimension ranging from about 0.5 micron×0.5 micron to about 20microns×20 microns in cross sectional area for a square-shaped probe.

In some embodiments, the apparatus may have typical channel dimensionsranging from about 6 microns×6 microns to about 14 microns×14 microns incross sectional area for a square-shaped channel, a radius ranging fromabout 3 microns to about 8 microns in cross sectional area for acircular shaped channel, and a typical probe dimension ranging fromabout 0.5 micron×0.5 micron to about 10 microns×10 microns in crosssectional area for a square shaped probe.

In some embodiments, the first material and the fourth material eachcomprise un-doped oxide (SiO₂), silicon nitride, doped oxide, a polymermaterial, glass, or an insulating material. Optionally, the abovementioned materials can be coated with at least one coating material forimproving compatibility (between biological sample and the surface ofthe apparatus in contact with the biological sample), easiness to clean,and apparatus reliability and lifetime.

In some embodiments, the second material and third material eachcomprise an electrically conductive material, aluminum, an aluminumalloy, copper, a copper alloy, tungsten, a tungsten alloy, gold, a goldalloy, silver, a silver alloy, conductive polymer, carbon nano-tube or apiezo-electrical material. Examples of the piezo-electrical materialinclude, but are not limited to, quartz, berlinite, gallium,orthophosphate, GaPO₄, tourmaline, ceramics, barium, titanate, BatiO₃,lead zirconate, titanate PZT, zinc oxide, aluminum nitride, and apolyvinylidene fluoride.

In yet some other embodiments, the second material and fourth materialeach comprise an electrically conductive material or a piezo-electricalmaterial. Examples of the electrically conductive material include, butare not limited to, aluminum, an aluminum alloy, copper, a copper alloy,tungsten, a tungsten alloy, gold, a gold alloy, silver, a silver alloy;whereas examples of the piezo-electrical material include, but are notlimited to, quartz, berlinite, gallium, orthophosphate, GaPO₄,tourmaline, ceramics, barium, titanate, BatiO₃, lead zirconate, titanatePZT, zinc oxide, aluminum nitride, and a polyvinylidene fluoride.

In some embodiments of the apparatus, the detection device comprises atleast one probe, at least one detector, or at least one pair of probeand detector, the probe generates a probing or disturbing signal ontothe biological subject to give a response signal, and the detectormeasures the response signal thus generated.

In some embodiments of the apparatus, the second material is patternedby microelectronic processes to form a first desired feature; the firstmaterial and third material are optionally patterned by microelectronicprocesses to form a second desired feature; and the first material andthird material can be the same or different.

In some embodiments, the methods for fabricating the apparatus furtherinclude capping the top of the material stack to form an enclosedtrench, with such trench used for test sample transportation ordetection site.

One of the key novel aspects of this patent application is the designand fabrication process flows of micro-devices and methods of using themicro-devices for contacting and measuring properties, at microscopiclevels and in a three dimensional space, of a biological subject (e.g.,a single cell or a single biological molecule such as DNA or RNA). Themicro-devices have micro-probes arranged in a three dimensional mannerwith feature sizes as small as a cell, a DNA, and a RNA and capable oftrapping, sorting, probing, measuring, communicating, moving,contacting, slicing, cutting, manipulating, or modifying biologicalsubjects.

Another aspect of this invention relates to methods for fabricating amicro-device. The methods include depositing various materials on asubstrate and, in the interims of depositing every two materials,pattern the materials by microelectronic technology and associatedprocesses, wherein the micro-device is capable of measuring at themicroscopic level the electrical, magnetic, electromagnetic, thermal,optical, photo-electrical, piezo-electrical, piezo-photonic,piezo-electrical photronic, acoustical, biological, mechanical,chemical, physical, physical-chemical, bio-chemical, bio-physical,bio-mechanical, bio-electrical, bio-thermal, bio-optical, bio-chemicalmechanical, bio-electro-mechanical, bio-electro-chemical mechanical,electro-chemical mechanical, micro-electro-mechanical property, or acombination thereof, of a biologic subject that the micro-device is tocontact.

Still another aspect of this invention relates to methods forfabricating a micro-device, which include depositing a first material onthe substrate, pattering the first material by a microelectronic processto give rise to at least one patterned residual and leaving part of thesubstrate surface uncovered by the first material, depositing a secondnon-conductive material atop the processed first material and thesubstrate, creating an opening in the second material and exposing partof the patterned residual of the first material, filling up the openingin the second material with a third material. In some embodiments, themicroelectronic process comprises thin film deposition,photolithography, etching, cleaning, or chemical mechanical polishing.

Yet in still another aspect, the invention provides methods forfabricating a micro-device, which include the first step of depositing afirst material onto a substrate; the second step of depositing a secondmaterial onto the first material and then patterning the second materialwith a microelectronic technology or process; and repeating the secondstep at least once with a material that can be the same as or differentfrom the first or second material. The materials used in the repeatedsteps can be the same as or different from the first or second material.In some embodiments, at least one of the materials used in fabricatingthe micro-device is a piezo-electrical material or a conductivematerial.

In some embodiments, multiple fabricated micro-devices can be coupled,joined, connected, and integrated by physical or electrical method toconstitute the more advanced devices.

In some embodiments, fabrication is done by a microelectronic process(e.g., chemical vapor deposition, physical vapor deposition, or atomiclayer deposition to deposit various materials on a substrate as aninsulator or conductor or semiconductor; lithography and etch orchemical mechanical polishing to transfer patterns from design tostructure; chemical mechanical planarization, chemical cleaning forparticle removal; thermal spiking anneal to reduce the crystal defects;diffusion or ion implantation for doping elements into specific layers).In some embodiments, patterning is planarization by chemical polishing,mechanical polishing, or chemical mechanical polishing.

In some other embodiments, the methods further include removal of astack of multiple layers of materials by wet etch, plasma etch, or byvapor etch.

In some embodiments, the micro-device can move in any direction. Forinstance, two micro-devices can move in opposite directions.

In some embodiments, the micro-device thus fabricated is so patternedthat it is capable of trapping, sorting, probing, measuring,communicating, manipulating, contacting, moving, slicing, cutting, ormodifying a biological subject; or that it can piece through themembrane of a cell.

Still another aspect of the invention relates to methods for fabricatinga device or apparatus for detecting disease in a biological subject,which include providing a substrate, sequentially depositing a firstmaterial and a second material as two different layers onto thesubstrate to form a material stack, patterning the second material bymicroelectronic processes to form a first desired feature, depositing athird material onto the material stack, optionally patterning the firstand third materials by microelectronic processes to form a seconddesired feature, and optionally depositing a fourth material onto thematerial stack. One, some, or all of the above processes and flows canbe repeated to form additional identical, variations, or differentstructures including but not limited to channels to transport biologicalsample, chambers for processing, treating, or measuring biologicalsamples, probers, detectors and other components.

In some embodiments, the methods further include steps of fabricating(utilizing processes including but not limited to depositing,patterning, polishing, and cleaning) additional components onto thesubstrate before sequentially depositing the first material and thesecond material as layers onto the substrate, wherein the additionalcomponents comprise a data storage component, a signal processingcomponent, a memory storage component, a signal transmitting component(receiving and sending signals), a logic processing component, or an RF(radio-frequency) component.

In some other embodiments, the methods further include steps offabricating at least a circuit onto the substrate before sequentiallydepositing the first material and the second material as layers onto thesubstrate, wherein the circuit comprises a data storage circuit, asignal processing circuit, a memory storage circuit, a signaltransmitting circuit, or a logic processing circuit.

In still some other embodiments, the methods of this invention furtherinclude a step of planarizing the third material using chemicalmechanical polishing process or an etch back process, after the step ofdepositing the third material onto the material stack and before thestep of patterning the first and the third materials.

Examples of the suitable microelectronic processes include, but are notlimited to, thin film deposition, lithography, etch, polishing,cleaning, ion implantation, diffusion, annealing, and packaging astypically used in microelectronics.

The first and third materials can be the same or different. They can be,for example, electrically insulating material, such as oxide, dopedoxide, silicon nitride, silicon carbide, aluminum oxide, or a polymer.

The second material can be an electrically conductive material, apiezo-electrical material, a piezo-photronic material, apiezo-electro-photronic material, a semiconductor material, a thermalsensitive material, an optical material, a pressure sensitive material,an ion emission sensitive material, or any combination thereof. Forexample, the second material can be copper, aluminum, tungsten, gold,silver, glass, an aluminum alloy, a copper alloy, a tungsten alloy, agold alloy, a silver alloy, quartz, berlinite, gallium, orthophosphate,GaPO₄, tourmaline, ceramics, barium, titanate, BatiO₃, lead zirconate,titanate PZT, zinc oxide, aluminum nitride, and a polyvinylidenefluoride.

In some embodiments, the first desired feature can be a probe, whereasthe second desired feature can be a recessed form, or a trench form inthe layers of the first and third materials.

In yet some other embodiment, the methods of this invention furthercomprise depositing a fourth material onto the material stack and thenpatterning the fourth material to form a recessed area such as a hole ata selected location. Further, optionally, additional materials andlayers can be added and processed to form additional features andcomponents.

In still another embodiment, the methods of this invention furthercomprise a step of removing the third material from the material stackby wet or vapor etch to form a detection chamber between the fourthmaterial and the substrate. Furthermore, they may also include a step ofremoving the first material from the material stack by wet etch or vaporetch to form a channel. The channel can connect the formed detectionchamber with additional chambers, and for transporting biologicalsamples.

In yet still another embodiment, the methods of this invention furtherinclude a step of sealing or capping the top of the material stack toform an enclosed trench. In one example of this step, the top of thematerial stack is sealed or capped with an additional device onto thematerial stack. Examples of such an additional device include, but arenot limited to, an imaging device, a communication device, and adetecting probe. The above said device on top of the material stackcomprises of optical device, imaging device, camera, viewing station,acoustic detector, piezo-electrical detector, piezo-photronic detector,piezo-electro photronic detector, electrical sensor, thermal detector,ion emission detector, and thermal recorder.

In another aspect, the invention provides micro-devices for detecting ortreating a disease, each comprising a first micro sensor for detecting aproperty of the biological sample at the microscopic level, and aninterior wall defining a channel, wherein the micro sensor is located inthe interior wall of the micro-device and detects the property of thebiological sample in the microscopic level, and the biological sample istransported within the channel. The size of the channel can range from0.5 micron to 80 microns in radius for a circular shaped channel, from 1micron to 50000 microns in length for each side for a rectangle shapedchannel. The property to be measured, e.g., can be an electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, bio-physical-mechanical, physical-chemical,electro-mechanical, electro-chemical, electro-optical, electro-thermal,electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-optical,bio-thermal, bio-physical, bio-electro-mechanical, bio-electro-chemical,bio-electro-optical, bio-electro-thermal, bio-mechanical-optical,bio-mechanical thermal, bio-thermal-optical,bio-electro-chemical-optical, bio-electro-mechanical-optical,bio-electro-thermal-optical, bio-electro-chemical-mechanical, physicalor mechanical property, or a combination thereof.

In some embodiments, the first micro sensor or micro-device isfabricated by microelectronics technologies. For example, the firstmicro sensor can be fabricated to be an integral part of an interiorwall of the micro-device, or the first micro sensor is fabricatedseparately from and bonded to the interior wall of the micro-device.

In some embodiments, each of the micro-devices may further comprise aread-out circuitry which is connected to the micro sensor and transfersdata from the first micro sensor to a recording device. The connectionbetween the read-out circuit and the first micro sensor is digital(e.g., with code or decoding technology), analog (e.g., through electronor proton movement or radio), optical, electrical, or mechanical (e.g.,with a nano-sized wire).

In some embodiments, each of the micro-devices may further comprises atleast one additional micro sensor in proximity with the first microsensor and located on the same interior wall, wherein the at least oneadditional micro sensor is fabricated in micro-technologies process. Forinstance, each micro-device may further comprises at least three (e.g.,5, 8, or 15) additional micro sensors in proximity with the first microsensor and located on the same interior wall as the first micro sensor,wherein the at least three additional micro sensors are fabricated inmicro-technologies process. These micro sensors can be arranged in onegroup or at least two groups (in a certain geometrical order).

In some embodiments, every two of the micro sensors can detect the sameor different properties of the biological sample, or they can performthe same or different functions. For example, at least one of the microsensors can be a probing sensor and apply a disturbing signal to thebiological sample, while at least another micro sensor only detects asignal or property at the microscopic level of the biological sample(whether or not it has been probed or disturbed by a probing sensor).

In some embodiments, the micro-sensors are fabricated on a flat paneland exposed to the channel defined by the interior walls of themicro-device.

In some embodiments, each micro-device of this invention has a symmetricinterior or exterior configuration. For example, the micro-device canhave an oval, circular, hexagon, triangular, square, or rectangularinterior configuration or channel.

In some embodiments, a micro-device of this invention has a square,oval, circular, hexagon, triangular, or rectangular interior channel andfour sides of interior walls. In some of these embodiments, all themicro sensors can be located on one side or two opposite sides of theinterior wall.

In some embodiments, a micro-device of this invention comprises twopanels, at least one of the panels is fabricated by micro-electronictechnologies and comprises the micro sensors and a read-out circuitry,with micro sensors located in the interior wall of the panel which withother interior walls of the micro-device defines the interior channel ofthe micro-device.

In some other embodiments, a micro-device of this invention furthercomprises two micro-cylinders that are placed between and bonded withthe two panels, wherein each of the micro-cylinders is solid, hollow, orporous, and optionally fabricated by microelectronics technologies. Forinstance, the micro-cylinders can be solid and at least one of themcomprises a micro sensor fabricated by microelectronics technologies.The micro sensor in the micro-cylinder can detect the same or differentproperty as a micro sensor in a panel of the micro-device. For example,a micro sensor in the micro-cylinder can be a probing sensor and appliesa probing or disturbing signal to the biological sample to be tested,whereas a micro sensor in a panel does not provide a disturbing signaland only detects a property of the biological sample at the microscopiclevel.

In some embodiments, at least one of the micro-cylinders comprises atleast two micro sensors fabricated by microelectronics technologies, andevery two of the at least two micro sensors are so located in thecylinder that an array of micro sensors in a panel at position betweenevery two micro sensors in the micro cylinder. For example, at least oneof the panels comprises at least two micro sensors that are arranged inat least two arrays each separated by at least a micro sensor in acylinder. Alternatively, at least one array of the micro sensors in thepanel can comprise two or more (e.g., 4, 9, or 16) micro sensors.

The two sensors in the micro cylinder can be apart by a distance rangingfrom 0.1 micron to 500 microns, from 0.1 micron to 50 microns, from 1micron to 100 microns, from 2.5 microns to 100 microns, from 5 micronsto 250 microns.

In some embodiments, a micro-device of this invention comprises twopanels each comprising at least one micro sensor and a read-outcircuitry, the micro-sensors are located in the interior wall of eachpanel which with other interior walls of the micro-device defines theinterior channel of the micro-device. For example, each panel maycomprise at least two micro sensors arranged in an array.

The micro-device may further comprise two micro-cylinders that areplaced between and bonded with the two panels, wherein each of themicro-cylinders can be solid, hollow, or porous, and optionallyfabricated by microelectronics technologies. For example, themicro-cylinders can be solid and at least one of them comprises a microsensor fabricated by microelectronics technologies.

The micro sensor in the micro-cylinder can detect the same or differentproperty as a micro sensor in a panel of the micro-device. For example,a micro sensor in the micro-cylinder can apply a probing signal to thebiological sample to be tested and cause the biological sample torespond by generating a signal.

In some other embodiments, at least one of the micro-cylinders comprisesat least two micro sensors fabricated by microelectronics technologies,and every two of the at least two micro sensors are so located in thecylinder that an array of micro sensors in a panel at position betweenthe every two micro sensors in the micro cylinder.

In some other embodiments, at least one of the panels comprises at leasttwo micro sensors that are arranged in at least two arrays eachseparated by at least a micro sensor in a cylinder. In some of theseembodiments, at least one array of the micro sensors in the panelcomprises two or more micro sensors.

In some further embodiments, each micro-device of this inventioncomprises:

two panels at least one of which is fabricated by microelectronicstechnologies and comprises the micro sensors and a read-out circuitry,and the micro sensors are located in the interior wall of the panelwhich, with other interior walls of the micro-device, defines theinterior channel of the micro-device;

two micro-cylinders that are placed between and bonded with the twopanels, wherein each of the micro-cylinders is solid, hollow, or porous,and optionally fabricated by microelectronics technologies; and

an application specific integrated circuit chip which is internallybonded to or integrated into one of the panels or a micro-cylinder and,together with other components of the micro-device defines the internalchannel of the micro-device.

In these embodiments, a micro-device may further comprises an opticaldevice, a piezo-electrical detector, a piezo-photronic detector, apiezo-electro photronic detector, an electrical detector, imagingdevice, camera, viewing station, acoustic detector, thermal detector,ion emission detector, or thermal recorder, each of which is integratedinto the a panel or a micro cylinder.

Each micro sensor can be a thermal sensor, an electrical sensor, anelectro-magnetic sensor, piezo-electrical sensor, piezo-photronicsensor, piezo-optical electronic sensor, image sensor, optical sensor,radiation sensor, mechanical sensor, magnetic sensor, bio-sensor,chemical sensor, bio-chemical sensor, or acoustic sensor.

Examples of micro sensor include a thermal sensor, piezo-electricalsensor, piezo-photronic sensor, piezo-optical electronic sensor, anelectrical sensor, an electro-magnetic sensor, image sensor, opticalsensor, radiation sensor, mechanical sensor, magnetic sensor,bio-sensor, chemical sensor, bio-chemical sensor, and acoustic sensor.Examples of thermal sensor comprise a resistive temperaturemicro-sensor, a micro-thermocouple, a thermo-diode andthermo-transistor, and a surface acoustic wave (SAW) temperature sensor.Examples of an image sensor include a charge coupled device (CCD) and aCMOS image sensor (CIS). Examples of a radiation sensor include aphotoconductive device, a photovoltaic device, a pyro-electrical device,or a micro-antenna. Examples of a mechanical sensor comprise a pressuremicro-sensor, micro-accelerometer, micro-gyrometer, and microflow-sensor. Examples of a magnetic sensor comprise a magneto-galvanicmicro-sensor, a magneto-resistive sensor, a magneto-diode, andmagneto-transistor. Examples of a bio-chemical sensor include aconductive-metric device and a potentio-metric device.

In still some other embodiments, a micro-device of this invention mayfurther include a read-out device for receiving or transferring datacollected by the micro sensor on the measured property of the biologicalsample.

Associated with the micro-devices described above are methods forfabricating a micro-device for detecting or treating a disease. Each ofthe methods can include the steps of: fabricating a first panel bymicroelectronics technologies, fabricating at least one micro sensor bymicroelectronics technologies and integrating it to the first panel,optionally providing or fabricating at least one micro-cylinder and asecond panel, bonding the first panel and the optional second panel andthe optional micro-cylinder whereby the interior walls of the panels andoptional micro-cylinder define an internal channel of the micro-deviceand the micro sensor is exposed in the internal channel. In someexamples of these methods, the at least one micro sensor is fabricatedas an internal part of and at the same time as the first panel. In someother examples, fabricating the first panel also gives rise to aread-out circuitry which is connected to the micro sensors in a panel bya digital, analog, or mechanical means.

Also associated with the micro-devices described above are methods fordetecting a disease in a subject in need thereof, each comprising thesteps of: taking a biological sample from the subject and taking abiological sample from disease-free subject, analyzing the twobiological samples to measure a property thereof at the microscopiclevel with a micro-device of this invention and comparing the measuredproperty of the two biological samples. The property to be measured canbe, e.g., an electrical, magnetic, electromagnetic, piezo-electrical,piezo-photronic, piezo-electro photronic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-physical,bio-thermal, bio-optical, bio-electro-mechanical, bio-electro-chemical,bio-electro-physical, bio-electro-thermal, bio-electro-optical,bio-electro-chemical-mechanical, physical, or mechanic property, or acombination thereof, of the biological sample.

In yet another aspect, the present invention provides methods forfabricating a device for detecting disease in a biological subject,which include providing a substrate, sequentially depositing a first anda second materials as layers onto the substrate to form a materialstack, patterning the second material, optionally by lithography andetch processes or by direct-writing process, to form a recessed area inthe layer of the second material, depositing a third material onto thematerial stack, removing a portion of the third material above thesecond material by etching back and/or polishing process (etching back,etching back followed by polishing, or by polishing process), patterningthe third material, optionally by lithography and etch processes or bydirect-writing process, to form at least a portion of recessed area inthe layer of the third material, depositing a fourth material onto thematerial stack, and removing the portion of the fourth material abovethe third material by etch back or polishing process to keep at least aportion of the second and fourth material in the same layer.

If desired, more layers of different materials can be deposited,patterned, cleaned, or planarized to form additional structures withmore features, components, layers, functionalities, and complexities.

The first and third materials used in the methods of this invention canbe the same or different. In some embodiments, they are the same. Theycan be, e.g., an electrically insulating material. Examples of the firstand third materials include, but are not limited to, oxide, doped oxide,silicon nitride, or a polymer.

In some embodiments, following the deposition and processing of thethird or fourth material, at least one more material is deposited andprocessed to form a top layer with a detection chamber or channelsformed underneath.

Examples of the second material include, but are not limited to,electrically conductive materials, piezo-electrical materials,piezo-electro photronic materials, semiconductor materials, thermalsensitive materials, a pressure sensitive material, an ion emissionsensitive material, optical materials, or any combinations thereof.

In some embodiments, a novel detection apparatus comprising a detectionchamber and/or channels for test sample transport is formed by methodsthat include the steps of: depositing a first material, patterning thefirst material (“material A”) to form at least a recessed area,depositing a second material (“material B”), removing the secondmaterial (“material B”) from areas above the first material (“materialA”) by using polishing and/or etch back processes, leaving the secondmaterial (“material B”) in the recessed area in the first materiallayer, depositing a third material (“material C”) to cover the firstmaterial (“material A”) and the second material (“material B”),patterning the third material (“material C”) to form at least a holesmaller than the recessed area(s) in the third material layer and aboveit, removing the second material (“material B”) optionally by usingvapor etch or wet etch or heating, forming an enclosed cavity in thefirst material layer.

In addition to novel micro-devices and manufacturing process forfabricating them, packaging of such devices are also critical (a) inensuring its proper function and (b) how to incorporate (transport itinto the micro-device) biological sample into the micro-device forprocessing, sorting, detection, probing, communicating, and possiblymanipulating, modifying and treating such biological subjects.Specifically, after being fabricated, the micro-devices typically needto be packaged for protection from outside environment and forconfiguration for connection with the outside world (e.g., by electricalconnection).

In this application, a set of novel designs, configurations, processes,and materials are disclosed, with the goals of protecting themicro-devices, connecting to the outside world, and transportingbiological samples into the micro-devices properly and effectively. Insome embodiments relating to this aspect, after being fabricated, amicro-device can be wrapped with a packaging material that forms aprotective or packaging layer around the micro-device. The packagingprocess may also allow for forming lead pins on the packaging materialfor connections (e.g., magnetic or electrical connection) with outsidedevices, e.g., for data transmissions and instruction communications.The packaging material can be an organic polymeric material, aninorganic polymeric material, or a molding compound.

In some other embodiments, a novel cavity can be formed in the packagingor protective layer, which has at least one opening connecting to theinlet of the micro-device and at least one other opening connecting toan outside device such as an injection device. In this way, a biologicalsample can be injected into the cavity through the opening (e.g., byconnecting to an injector) and transported into the micro-device throughthe other opening connecting to the micro-device inlet.

In still some other applications, an outside device such as an injectiondevice can be directly connected to an inlet of, or fitted into, themicro-device for transporting a biological sample. In this case, it isimportant that the inlet is leak free at both ends connected to themicro-device and to the outside device (such as an injector). To achievethis, a first material with substantially high viscosity can be usedfirst to seal seams and cracks between the inlet and the micro-device,or between the outside device and the micro-device. It could be a solidmaterial or a material with very high viscosity. To secure its stabilityand resolve possible adhesion issues with the first material and thedevice, a second material (e.g., a material that has a lower viscosityand is sticky in nature, when melt or solution) can be applied. Examplesof such a material include epoxies, adhesives, and glues. To speed upthe drying process of the second material when it is in a solution, heatcan be applied (for example, an air flow at a temperature of 40° C. orhigher).

In yet some other embodiments, a novel detection apparatus can beintegrated with at least one micro-injector and at least one detector,in which the micro-injector can inject a desired object into thebiological subject to be tested to generate a response by the biologicalsubject and the detector detects the response thus generated by thebiological subject.

The invention further provides methods for detecting a biologicalsubject's dynamic response to a signal. These methods include providingan apparatus comprising two micro-devices of which one is a probingmicro-device and the other is a detecting micro-device and positionedwith a distance from the probing micro-device; contacting the biologicalsubject with the probing micro-device whereby the probing micro-devicemeasures a property of the biological subject at the microscopic levelor sends a stimulating (disturbing) signal to the biological subject;and the detecting micro-device measures the response of the biologicalsubject through measuring properties of the biological subject at themicroscopic level. Optionally, the detecting micro-device contacts thebiological subject during the measurements. How the above statedstimulating (disturbing) signal is applied (for example, the speed withwhich it is applied) and its magnitude can be important to obtain thebest and/or largest response from the biological sample being tested.For example, when a thermal wave is used as a stimulating (a disturbing)signal, how fast it ramps up from its initial value to its final value(for example, from 30° C. to 40° C.) could have important effects onmaximizing its response signal from the biological sample.

In some embodiments, the stimulating (a disturbing) or response signalis an electrical, magnetic, electromagnetic, piezo-electrical,piezo-photronic, piezo-electro photronic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-optical, electro-thermal, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-physical, bio-optical, bio-thermal,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical signal, or acombination thereof.

In some other embodiments, the property at the microscopic level is anelectrical, magnetic, electromagnetic, piezo-electrical,piezo-photronic, piezo-electro photronic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-optical, electro-thermal, electro-chemical-mechanical,bio-chemical, bio-chemical-physical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical, bio-physical, bio-optical,bio-thermal, bio-electro-chemical-mechanical, physical, or mechanicalproperty, or a combination thereof.

For both stimulating (a disturbing) and response signal, examples of theelectrical properties include, but are not limited to, surface charge,surface potential, resting potential, electrical current, electricalfield distribution, electrical dipole, electrical quadruple,three-dimensional electrical and/or charge cloud distribution,electrical properties at telomere of DNA and chromosome (also calledsticky end or DNA end) capacitance, or impedance. Examples of thethermal properties include temperature, and vibrational frequency ofbiological item and molecules. Examples of the optical propertiesinclude optical absorption, optical transmission, optical reflection,optical-electrical properties, brightness, and fluorescent emission.Examples of the chemical properties include pH value, chemical reaction,bio-chemical reaction, bio-electro-chemical reaction, reaction speed,reaction energy, speed of reaction, oxygen concentration, oxygenconsumption rate, ionic strength, catalytic behavior, added chemicalcomponents, and bonding strength. Examples of the physical propertiesinclude density, and geometric shape and size (volume and surface area).Examples of the acoustic properties include frequency, speed of acousticwaves, acoustic frequency and intensity spectrum distribution, acousticintensity, acoustical absorption, and acoustical resonance. Examples ofthe mechanical property include internal pressure, flow rate, viscosity,hardness, shear strength, elongation strength, fracture stress,adhesion, mechanical resonance frequency, elasticity, plasticity, andcompressibility. Examples of biological properties include a biologicalsubject's surface properties (such as surface shape, surface area,surface charge, and surface biological and chemical properties) andproperties of solutions in which a biological subject resides (such aspH, electrolyte, ionic strength, resistivity, cell concentration, andbiological, electrical, physical properties, and chemical properties).The data from measuring one or more of the properties at the microscopiclevel can be used for detecting diseases, e.g., cancer at its earlystage, or for estimating the life expectancy of the carrier of thebiological subject.

In some other embodiments, the apparatus further includes a thirdmicro-device that is different from the probing micro-device and thedetecting micro-device; and the third micro-device measures the same ora different property of the biological subject as the probingmicro-device and the detecting micro-device do.

In still some other embodiments, the apparatus further includes a clockmicro-device that is different from the probing micro-device and thedetecting micro-device; and the type of clock micro-device is placed ata fixed distance before the probing micro-devices and detectingmicro-devices with a distinctive signal when a biological subject passesit and acts as a clock device.

Yet still in some embodiments, the data recorded by the detectingmicro-device is filtered by a phase lock-in technology to remove noiseunsynchronized to the clock signal in order to enhance signal to noiseratio and improve measurement sensitivity. For example, in order toenhance measured response signal from the biological sample and reducenoise, a simulating (disturbing) signal can be in a pulsed form (forexample, a pulsed laser beam at a desired frequency) or an alternatingpattern (for example, an alternating current), and a lock-in amplifiercan be utilized to only amplify the part of the measured response signalwhich is synchronized to the frequency of the simulating (disturbing)signal.

Another aspect of this invention relates to methods for detectingdisease in a biological subject, comprising providing an apparatuscomprising a channel, a detection probe, imaging device, a memorystorage component, a signal transmitting component, a signal receivingcomponent, or a logic processing component, pre-processing thebiological subject to enhance its concentration, measuring theproperties of the biological subject, optionally contacting thebiological subject with the probing component (probing micro-device orprobing tip) through the channel to trigger or result in a responsesignal, using the detection probe (e.g., detection micro-device ordetection component) to detect the response signal from the biologicalsubject, optionally separating diseased biological subject from healthybiological subject based on the response signal, optionally sending theseparated, suspected diseased biological subject on for further tests,and analyzing the response signal and reaching a diagnosis conclusion.The biological subject can be a DNA, a sub-structure in a cell, a cell,or a protein.

In some embodiments, the methods of this invention further includedetection of the response signal and behaviors of interaction or eventsoccurred between at least two biological subjects or at least onebiological subject with at least one non-biological subject. The atleast two biological subject can be different or identical, in type ofcomposition. Examples of interactions or events occurred between atleast two biological subjects include, but are not limited to, a DNAcolliding with another DNA, a cell smashing into another cell, a DNAcrashing into a cell, a protein colliding with another protein, or a DNAcrashing into a protein. Examples of interactions or events occurredbetween at least one biological subject with at least one non-biologicalsubject include, but not limited to, an inorganic particle collidingwith a biological subject, an organic particle colliding with abiological subject, or a composite particle colliding with a biologicalsubject.

Examples of the response signals include, but are not limited to, anelectrical, magnetic, electromagnetic, thermal, optical,piezo-electrical, piezo-electro photronic, piezo-photronic, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-physical, electro-thermal, electro-optical,electro-chemical-mechanical, bio-chemical, bio-physical, bio-optical,bio-thermal, bio-electromagnetic, bio-chemical-physical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, and mechanical signal, or acombination thereof.

In some embodiments of the apparatus of this invention, the system fordelivering the biological subject includes at least one channel insidewhich the biological subject to be detected travels in a certaindirection; the probing and detecting device includes at least oneprobing micro-device and at least one detecting micro-device, at leastone probing micro-device is located before at least one detectingmicro-device relative to the direction in which the biological subjecttravels, and the probing micro-device and the detecting micro-device canbe attached to the interior or exterior wall of the channel. In someother embodiments, multiple channels with different geometries areutilized.

In some examples of these embodiments, the probing and detecting deviceincludes at least two detecting micro-devices capable of measuring atthe micro-level the same or different properties of the biologicalsubject.

In some other embodiments, the shapes and sizes of different sections ofthe channel can be the same or different; the width of the channel canbe about 1 nm˜1 mm (e.g., 1˜750 nm, 1˜600 nm; 100˜800 nm, 200˜750 nm, or400˜650 nm); the channel can be straight, curved, or angled; theinterior wall of the channel defines a circular, oval, or polygon (e.g.,rectangular) space.

An example of a suitable channel is a circular carbon nano-tube, whichcan have a diameter of, e.g., about 0.5˜100 nm, or a length of, e.g.,about 5.0 nm˜10 mm.

Another example of a suitable channel is a square channel in silicon orpolysilicon substrate, which can have a size (for each side) ranging,e.g., from 0.5 micron to 100 microns, and a length ranging, e.g., from5.0 nm to 10 mm.

Yet another example of a suitable channel is a square channel in silicondioxide substrate, which can have a size of (for each side), e.g., from0.5 micron to 100 microns, and a length of, e.g., about 5.0 nm˜10 mm.

Optionally, channels are coated with a thin film for enhancedcompatibility with biological subjects, and/or improved detectionsensitivity and/or efficiency.

In some embodiments, the interior wall of the channel has at least oneconcave that may be in the same section as a probing or detectingmicro-device. The concave groove can be, e.g., a cubic space or anangled space. It can have a depth of, e.g., about 10 nm˜1 mm.

In some other embodiments, a distribution fluid can be injected into thechannel, either before or after the biological subject passes a probingmicro-device, to aid the traveling or separation of the biologicalsubject inside the channel. A suitable distribution fluid is abiocompatible liquid or solution, e.g., water or saline. Thedistribution fluid can be injected into the channel through adistribution fluid channel connected to an opening in the channel wall.Utilizing such a distribution fluid allows, among others, preparation ofthe surface of the channel (in which the biological subject travels),cleaning of the channel, disinfection of the apparatus, and enhancingthe measurement sensitivity of the apparatus.

In yet some other embodiments, a cleaning fluid can be used to clean anapparatus of this invention, particularly narrow and small spaces in theapparatus wherein biological residues and deposits (e.g., dried blood orprotein when it is used as or contained in a sample to be tested by theapparatus) are likely to accumulate and block such spaces. Desiredproperties of such a cleaning fluid include low viscosity and ability todissolve the biological residues and deposits. For example, when anapparatus of this invention is used for detecting a disease, certainbiological samples, such as blood, could result in blockage to narrow,small spaces in the apparatus such as narrow channels when the blood isallowed to dry. The cleaning solution is expected to address this issueby dissolve the biological samples.

In still some other embodiments, the apparatus of this invention can befor detecting the diseases of more than one biological subject, and thechannel comprises a device located therein for separating or dividingthe biological subjects based on different levels of a same property ofthe biological subjects. An example of such a separating or dividingdevice is a slit that can, e.g., separate or divide biological subjectsbased on their surface charges, surface chemistry, surface biologicalfeatures and properties, their density, their size, or other propertiessuch as electrical, thermal, optical, chemical, physical, biological,acoustical, magnetic, electromagnetic, and mechanical properties.

In yet still some other embodiments, the apparatus of this invention canfurther include a filtering device for removing irrelevant objects fromthe biologic subject for detection.

In another aspect, the invention provides methods for obtaining dynamicinformation of a biologic material, each comprising contacting thebiological subject (e.g., including but not limited to a cell,substructure of a cell such as cell membrane, a DNA, a RNA, a protein,or a virus) with an apparatus comprising a first micro-device, a secondmicro-device, and a first substrate supporting the first micro-deviceand second micro-device, wherein the first micro-device is capable ofmeasuring at the microscopic level an electrical, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,physical, or mechanical property, or a combination thereof, of thebiological subject, and the second micro-device contacts the biologicalsubjects and stimulates it with a signal.

In yet another embodiment, the micro-device in the detection apparatuscan communicate with biological subjects such as cells, DNA, RNA, virus,tissue, or protein. Further, the micro-device can trap, sort, analyze,treat, and modify biological subjects such as cells, DNA, RNA, virus,tissue, or protein. Specifically, an array of micro-devices arranged ina desired manner can trap, sort, probe, detect, communicate, manipulate,move, contact, and modify DNA structures.

In some embodiments, the apparatus further comprising a thirdmicro-device that is capable of measuring at the microscopic level thesame electrical, magnetic, electromagnetic, thermal, optical,acoustical, biological, chemical, bio-chemical, bio-physical,bio-electrical, bio-mechanical, bio-optical, bio-thermal, bio-magnetic,bio-electromagnetic, bio-electro-mechanical, bio-electro-chemical,bio-electro-physical, bio-electro-optical, bio-electro-thermal,bio-chemical-mechanical, physical, or mechanical property, or acombination thereof, of the cell as the first micro-device is. In someother embodiments, the cell contacts the first micro-device, secondmicro-device, and third micro-device in the order. In still some otherembodiments, the signal is an electrical signal, a magnetic signal, anelectromagnetic signal, a thermal signal, an optical signal, anacoustical signal, a biological signal, a chemical signal, a physicalsignal, an electro-optical signal, an electro-chemical signal, anelectro-mechanical signal, a bio-chemical signal, or abio-chemical-mechanical signal.

In another aspect, this invention provides alternative methods fordetecting a biological subject's dynamic information. The methods eachinclude providing an apparatus comprising a clock micro-device, aprobing micro-device, and a first detection micro-device, with theprobing micro-device being placed between the clock micro-device and thedetection micro-device; contacting the biological subject with the clockmicro-device whereby the clock micro-device registers the arrival of thebiological subject, and optionally measures a property of the biologicalsubject at the microscopic level; contacting the biological subject withthe probe device with a periodic probe signal delivered onto thebiological subject; using the detecting micro-device to detect responsesignal from the biological subject; and processing the detected signalby the detection micro-device using phase lock-in technology to filterout signal components un-synchronized to the frequency of the probesignal, and amplify the signal synchronized to the probe signal.

In some embodiments of these methods, there is a distance of at least 10angstroms between the clock micro-device and the first detectingmicro-device.

In some other embodiments, the response signal is an electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-optical, electro-thermal,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-electrical, bio-optical, bio-thermal, bio-physical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical, bio-electro-optical,bio-electro-thermal, bio-electro-chemical-mechanical, physical, ormechanical signal, or a combination thereof.

In some other embodiments, the first probing micro-device optionallymeasures the same property of the biological subject at the microscopiclevel as the first detecting micro-device does.

In still some other embodiments, the apparatus used in the methodsfurther comprises a second probing micro-device capable of sending astimulating signal to the biological subject that is different from thesignal sent by the first probing micro-device.

In still some other embodiments, the apparatus used in the methodsfurther comprise a second detecting micro-device capable of measuringthe same property of the biological subject at the microscopic level asthe first detecting micro-device does.

In some embodiments, the data recorded by the first detectingmicro-device is filtered by a phase lock-in technology to remove noiseunsynchronized to the data recorded by the first probing micro-device orthe clock micro-device. The filtered data may have a higher signal tonoise ratio.

Still yet another aspect of this invention is the design, fabrication,and integration of the various components in the disease detectionapparatus. These components include, e.g., a sample containment anddelivery unit; an array of sample delivery channels; a central diseasedetection unit comprising multiple detection probes, a central controlunit comprising a logic processing unit, a memory unit, a sensor, asignal transmitter, a signal receiver, and an application specific chip;and a waste sample treatment unit in which used sample can be treated,recycled, processed for reuse, or disposed.

Another key novel aspect of the current application is the design,integration, and fabrication process flow of micro-devices capable ofmaking highly sensitive and advanced measurements on very weak signalsin biological systems for disease detection under complicatedenvironment with very weak signal and relatively high noise background.Those novel capabilities using the class of micro-devices disclosed inthis invention for disease detection include, e.g., making dynamicmeasurements, real time measurements (such as time of flightmeasurements, and combination of using probe signal and detectingresponse signal), phase lock-in technique to reduce background noise,and 4-point probe techniques to measure very weak signals, and uniqueand novel probes to measure various electronic, electromagnetic andmagnetic properties of biological samples at the single cell, biologicalsubject (e.g., virus) or molecule (e.g., DNA or RNA) level. For example,for making dynamic measurements to further enhance measurementsensitivity, during measurements, at least one of the parameters appliedto the biological sample being measured or at least one of theproperties in the surrounding media (in which the biological sampleresides) is intentionally changed from a static state (constant value)to a dynamic state (for example, a pulsed value or an alternatingvalue), or from one value to a new value. As a novel example, in ameasurement, a DC current applied to a biological sample isintentionally changed to an AC current. In another novel example, aconstant temperature applied to a biological sample is changed to ahigher temperature, or a pulsed heat wave (for example, from 30° C. to50° C., then from 50° C. back to 30° C.).

Finally, another aspect of this invention relates to apparatus fordetecting disease in a biological subject. The apparatus includes adetection device fabricated by a method comprising: providing asubstrate; sequentially depositing a first material and a secondmaterial as two layers onto the substrate to form a material stack;patterning the second material by microelectronics processes to form afirst desired feature; depositing a third material onto the materialstack to cover the second material; optionally patterning the first andthird materials by microelectronic processes to form a second desiredfeature; and optionally depositing a fourth material onto the materialstack. The first material and third material can be the same ordifferent. The detection device is capable of probing the biologicalsubject to be detected and giving rise to a response signal.

In some embodiments, the fabrication method further comprises cappingthe top of the material stack to form an enclosed trench.

In some other embodiments, the capping comprises sealing or capping thetop of the material stack with an imaging device onto the materialstack.

In still some other embodiments, the apparatus further includes apre-processing unit (chambers) for pre-screening and enhancing adiseased biological subject for further testing, channels for carryingfluidic sample to flow through, probes for probing and disturbing thebiological subject being tested for generating response signals,detection probes for measuring properties and response signals of thebiological subject, or an imaging device for observing and recordingproperties and behaviors of the biological subject.

In yet some other embodiments, the detection device has typical channeldimensions ranging from about 2 microns×2 microns to about 100microns×100 microns in cross sectional area for a square-shaped channel,a radius ranging from about 0.5 micron to about 80 microns in crosssectional area for a circular shaped channel, and a typical probedimension ranging from about 0.5 micron×0.5 micron to about 20microns×20 microns in cross sectional area for a square-shaped probe.Alternatively, the detection device has typical channel dimensionsranging from about 6 microns×6 microns to about 80 microns×80 microns incross sectional area for a square-shaped channel, a radius ranging fromabout 3 microns to about 60 microns in cross sectional area for acircular shaped channel, and a typical probe dimension ranging fromabout 0.5 micron×0.5 micron to about 10 microns×10 microns in crosssectional area for a square shaped probe.

In yet still some embodiments, the first and the fourth materials eachcomprise un-doped oxide (SiO₂), doped oxide, silicon nitride, a polymermaterial, glass, or an electrically insulating material; the second andthird materials each comprise an electrically conductive material,aluminum, an aluminum alloy, copper, a copper alloy, tungsten, atungsten alloy, gold, a gold alloy, silver, a silver alloy, an opticalmaterial, an thermal sensitive material, a magnetic material, anelectromagnetic material, an electro-optical material, a pressuresensitive material, a mechanical stress sensitive material, an ionemission sensitive material, a piezo-electrical material, apiezo-photronic material, a piezo-electro photronic material.

In yet still some other embodiments, where the second and fourthmaterials can be fabricated at the same level as detectors, or as probesand detectors, the first and the third materials each comprise un-dopedoxide (SiO₂), doped oxide, silicon nitride, silicon carbide, a polymermaterial, glass, or an electrically insulating material; the second andfourth materials each comprise an electrically conductive material(e.g., aluminum, an aluminum alloy, copper, a copper alloy, tungsten, atungsten alloy, gold, a gold alloy, silver, or a silver alloy,refractory metals, carbon nano-tube), an optical material (e.g.,anisotropic optical material, glass, glass-ceramic, laser gain media,nonlinear optical material, fluorescent materials, phosphor andscintillator, transparent material), an thermal sensitive material, amagnetic material, an electromagnetic materials, a pressure sensitivematerial, a mechanical stress sensitive material, an ion emissionsensitive material, and a piezo-electrical material (e.g., quartz,berlinite, gallium, orthophosphate, GaPO₄, tourmaline, ceramics, barium,titanate, BatiO₃, lead zirconate, titanate PZT, zinc oxide, aluminumnitride, a polyvinylidene fluoride), piezo-photronic materials,piezo-electro photronic materials, electro-optical materials,electro-thermal materials.

In further embodiments, the detection device comprises at least oneprobe, at least one detector, at least one pair of probe and detector inwhich the probe generates a probing or disturbing (stimulating) signalonto the biological subject to give a response signal and the detectormeasures the response signal thus generated.

In other aspects, the present invention provides methods for fabricatingmicro-devices or micro-detectors of this invention by microelectronicprocess which may include deposition, lithography, etch, cleaning,direct writing, molecular self assembly, laser oblation, electron beamwriting, x-ray writing, diffusion, annealing, ion implantation,cleaning, polishing, planarization, or packaging.

In some embodiments, the methods fabricating a micro-device ormicro-detector include depositing various materials on a substrate and,in the interims of depositing every two materials, patterning some orall of the deposited materials by a microelectronic process.

In some other embodiments, the fabrication methods each include thesteps of:

providing a substrate;

depositing a first material onto the substrate;

depositing a second material onto the first material and then patterningthe second material by a microelectronic process; and

repeating the second step at least once with a material that can be thesame as or different from any of the previously deposited materials.

The methods may further include removal of a stack of multiple layers ofmaterials by wet etch, dry etch, vapor etch, direct writing, oblationsuch as laser oblation, or selective removal (for example, using localheating, local bombardment by ions, or localized sonic energy).

In these methods, the materials used in the repeated steps can be thesame as or different from the first or second material. At least one ofthe materials used in fabricating the micro-device is a biologicalmaterial, a bio-chemical material, a bio-inorganic compound material, apolymer, a piezo-electrical material, a piezo-photronic material, apiezo-electro photronic material, a thermal material, an opticalmaterial, an electro-optical material, a semiconductor material, anelectrically insulating material, or an electrically conductivematerial.

The micro-device thus fabricated can have one or more characters orfunctions of the following: moving in any direction; being capable ofsorting, probing, measuring, detecting, manipulating, moving, cutting,slicing, communicating, or modifying a biological subject.

Still, the methods may further include one or more of the followingsteps:

depositing a third material on the second material and then patterningthe third material by a planarization process;

depositing a fourth material on the third material and patterning thefourth material by microelectronic processes;

patterning the third material using a microelectronic process with thefourth material serving as a hardmask;

coupling two devices that are thus fabricated and symmetric to form adetecting device with channels or to form a probing device capable tosending a signal to a biological subject and resulting in a response;

integrating three or more micro-devices to give an enhanced device withan array of the channels.

Still further, the methods may include the steps of:

before depositing the second material, patterning the first material bya microelectronic process to give rise to at least one patternedresidual and leaving part of the substrate surface uncovered by thefirst material;

creating an opening in the second material to expose part of thepatterned residual of the first material;

filling up the opening in the second material with a third material;wherein the second material is a non-electrically conductive material;

optionally planarizing the third material, with third material remainingin the recessed area of the second material;

optionally depositing a fourth material;

optionally creating an opening in the fourth material;

optionally selectively removing substantially the third material, withthe first material, the second material, and the fourth materialsubstantially remaining; and

optionally sealing the opening in the fourth material by depositing afifth material.

In the above process flow, planarization of the third material can becarried out by etching back, etching back followed by polishing, orpolishing process. In addition, the removal of the third materialfollowing the deposition of the fourth material can be carried out usingwet etching, vapor etching, or heating (if evaporation temperature ofthe third material is higher than those of the other materials).

The micro-device thus obtained may include a micro-trench (or channel)having side-walls and a probe embedded in the micro-trench or channel'ssidewalls. Each channel's entrance may be optionally bell-mouthed; theshape of each channel's cross-section is rectangle, ellipse, circle,oval, or polygon. The dimension of the micro-trench may range from about0.1 um to about 500 um.

The micro-trench of the micro-device can be capped with a flat panel orcoupling two micro-trenches to form one or more channels. The flat panelmay comprise silicon, SiGe, SiO₂, Al₂O₃, acrylate polymer, AgInSbTe,synthetic alexandrite, arsenic triselenide, arsenic trisulfide, bariumfluoride, CR-39, cadmium selenide, caesium cadmium chloride, calcite,calcium fluoride, chalcogenide glass, gallium phosphide, GeSbTe,germanium, germanium dioxide, glass code, hydrogen silsesquioxane,Iceland spar, liquid crystal, lithium fluoride, lumicera, METATOY,magnesium fluoride, magnesium oxide, negative index metamaterials,neutron super mirror, phosphor, picarin, poly(methyl methacrylate),polycarbonate, potassium bromide, sapphire, scotophor, spectralon,speculum metal, split-ring resonator, strontium fluoride, yttriumaluminum garnet, yttrium lithium fluoride, yttrium orthovanadate, ZBLAN,zinc selenide, zinc sulfide, fluorescent materials, phosphorousmaterials, or electro-optical materials.

In some other embodiments, the methods for fabricating a micro-device ormicro-detector of this invention include the steps of:

providing a substrate;

sequentially depositing a first material and a second material as twolayers onto the substrate to form a material stack;

patterning the second material by microelectronic processes to form afirst desired feature; depositing a third material onto the materialstack;

optionally patterning the first and third materials by microelectronicprocesses to form a second desired feature; and

optionally depositing a fourth material onto the material stack.

They may further include:

fabricating at least an additional component onto the substrate beforesequentially depositing the first material and the second material aslayers onto the substrate, wherein the additional component comprises adata storage component, a signal processing component, a memory storagecomponent, a signal receiver, a signal transmitting component, a logicprocessing component, a data decoder, an application specific chipcomponent, or an RF component; or

fabricating at least an integrated circuit onto the substrate beforesequentially depositing the first material and the second material aslayers onto the substrate, wherein the integrated circuit comprises adata storage circuit, a signal processing circuit, a memory storagecircuit, a signal transmitting circuit, a sensor, or a logic processingcircuit. Alternatively, the above mentioned components (the additionalcomponent comprises a data storage component, a signal processingcomponent, a memory storage component, a signal receiver, a signaltransmitting component, a logic processing component, a data decoder, anapplication specific chip component, or an RF component) can befabricated on a separate substrate as a chip and it then can be bondedwith or integrated with the substrate containing the material stack(which comprises chambers, channels, and detection components). This canbe accomplished utilizing such technologies as flip chip, wafer bonding,and Through Silicon Via (TSV) technologies.

In some instances, the first material and the third material are thesame; the first material and the third material are electricallyinsulating (e.g., an oxide, doped oxide, silicon nitride, siliconcarbide, or a polymer); the first material and the fourth material arethe same; the first material and the fourth material are electronicallyinsulating; the second material or the third material is an electricalconductive material, a magnetic material, an electro-magnetic material,an optical material, a thermal sensitive material, a pressure sensitivematerial, an ion emission sensitive material, a piezo-electricalmaterial, piezo-electro photronic material, piezo-photronic material, anelectro-optical material, an electro-thermal material, a bio-chemicalmaterial, a bio-mechanical material, or a bio-inorganic material.

In some other instances, the second material is an electricallyconductive material, a piezo-electrical material, a piezo-electricalmaterial, piezo-electro photronic material, piezo-photronic material, anelectro-optical material, an electro-thermal material, a bio-chemicalmaterial, a bio-mechanical material, a bio-inorganic material, asemiconductor material, a thermal sensitive material, a magneticmaterial, a pressure sensitive material, a mechanical stress sensitivematerial, an ion emission sensitive material, an optical material, or acombination thereof. For example, it may include copper, aluminum,tungsten, gold, silver, refractive metals, fluorescent materials,phosphorous materials, the alloys thereof, or glass.

The detector thus fabricated may be capable of probing or disturbing(simulating) a biological subject to be measured; and it may have arecessed form, or a trench form in the layers of the third and firstmaterials. In the detector, the second material may be aligned with thewall of the trench form in the layers of the third and first materials.

In some instances, the methods may further include the step of cappingthe top of the material stack to cover the third material and form anenclosed trench. As an example, the capping may include sealing orcapping the top of the material stack with a layer of material, animaging device, a camera, a viewing station, an acoustic detector, athermal detector, an ion emission detector, a piezo-electrical detector,a piezo-photronic detector, a piezo-electro photronic detector, anelectro-optical detector, or a thermal recorder onto the material stack.

In some other instances, the methods may still further include one ormore of the following steps:

fabricating at least one integrated circuit onto the substrate beforesequentially depositing the first material and the second material aslayers onto the substrate, wherein the circuit comprises a data storagecircuit, a signal processing circuit, a memory storage circuit, asensor, a signal transmitting circuit, a sensor, or a logic processingcircuit;

planarizing the third material using a chemical mechanical polishingprocess or an etch back process after depositing the third material ontothe material stack and before patterning the first and the thirdmaterials;

planarizing the third material using a chemical mechanical polishingprocess or an etch back process to form a detector capable of detectinga response signal from the biological subject;

patterning the fourth material to form a hole at a selected locationafter depositing the fourth material onto the material stack;

removing the third material from the material stack by wet or vapor etchto form a detection chamber between the fourth material and thesubstrate;

removing the first material from the material stack by wet etch or vaporetch or heating to form a channel;

capping the top of the material stack to form an enclosed trench orchannel;

sealing or capping the top of the material stack with a fifth materialto form an enclosed channel capable of observing and recording thebiological subject; or

sealing or capping the top of the material stack with an imaging device,a detector, an optical sensor, a camera, a viewing station, an acousticdetector, a thermal detector, an electrical detector, an ion emissiondetector, a piezo-electrical detector, a piezo-photronic detector, apiezo-electro photronic detector, an electro-optical detector, or athermal recorder onto the material stack.

In still some embodiments, the methods for fabricating a micro-device ofthis invention include the steps of:

providing a substrate;

sequentially depositing a first material and a second material as layersonto the substrate to form a material stack;

patterning the second material by microelectronic processes to form atleast a portion of a recessed area in the second material (e.g., to forma probe, a detector or an integrated unit with sub-component fordetection);

depositing a third material onto the material stack to cover the secondmaterial, and removing the portion of the third material above thesecond material by etch back or polishing process;

patterning the third material by lithography and etch processes toremove at least a portion of the third material;

depositing a fourth material onto the material stack to cover the secondand third material, and removing the portion of the fourth materialabove the second and third material by etch back or polishing process;and

optionally, depositing a fifth material and repeating the above processsequence used for the third material.

In some instances, they may further include one or more steps of thefollowing:

fabricating at least an additional component onto the substrate beforesequentially depositing the first material and the second material aslayers onto the substrate, wherein the additional component comprises adata storage component, a signal processing component, a memory storagecomponent, a signal transmitting component, a logic processingcomponent, a data decoder, an application specific chip component, or anRF component; and

fabricating at least one integrated circuit onto the substrate beforesequentially depositing the first material and the second material aslayers onto the substrate, wherein the integrated circuit comprises adata storage circuit, a signal processing circuit, a memory storagecircuit, a signal transmitting circuit, a sensor, a data decoder, anapplication specific chip component, or a logic processing circuit.

The substrate can be silicon, polysilicon, silicon nitride, or polymermaterial; the first material is oxide, doped oxide, silicon nitride,silicon carbide, or polymer material. The second and the fourthmaterials can be the same (e.g., both being an electrical conductivematerial, semiconductor material, piezo-electrical material,piezo-electro photronic material, piezo-photronic material, anelectro-optical material, an electro-thermal material, a bio-chemicalmaterial, a bio-mechanical material, a bio-inorganic material, thermalsensitive material, an ion emission sensitive material, a magneticmaterial, a pressure sensitive material, a mechanical stress sensitivematerial, or optical material). Specific examples of suitable materialsinclude aluminum, copper, tungsten, gold, silver, refractory metals, thealloys thereof, quartz, berlinite, gallium, orthophosphate, GaPO₄,tourmalines, ceramics, barium, titanate, BatiO₃, lead zirconate,titanate PZT, zinc oxide, aluminum nitride, polyvinylidene fluoride,fluorescent materials, phosphorous materials, electro-optical materials,bio-optical materials, bio-electro optical materials.

As used herein, the term “or” is meant to include both “and” and “or”.It may be interchanged with “and/or.”

As used herein, a singular noun is meant to include its plural meaning.For instance, a micro device can mean either a single micro device ormultiple micro-devices.

As used herein, the term “patterning” means shaping a material into acertain physical form or pattern, including a plane (in which case“patterning” would also mean “planarization.”)

As used herein, the term “a biocompatible material” refers to a materialthat is intended to interface with a living organism or a living tissueand can function in intimate contact therewith. When used as a coating,it reduces the adverse reaction a living organism or a living tissue hasagainst the material to be coated, e.g., reducing the severity or eveneliminating the rejection reaction by the living organism or livingtissue. As used herein, it encompasses both synthetic materials andnaturally occurring materials. Synthetic materials usually includebiocompatible polymers, made either from synthetic or natural startingmaterials, whereas naturally occurring biocompatible materials include,e.g., proteins or tissues.

As used herein, the term “a biological subject” or “a biological sample”for analysis or test or diagnosis refers to the subject to be analyzedby a disease detection apparatus. It can be a single cell, a singlebiological molecular (e.g., DNA, RNA, or protein), a single biologicalsubject (e.g., a single cell or virus), any other sufficiently smallunit or fundamental biological composition, or a sample of a subject'sorgan or tissue that may having a disease or disorder.

As used herein, the term “disease” is interchangeable with the term“disorder” and generally refers to any abnormal microscopic property orcondition (e.g., a physical condition) of a biological subject (e.g., amammal or biological species).

As used herein, the term “subject” generally refers to a mammal, e.g., ahuman person.

As used herein, the term “microscopic level” refers to the subject beinganalyzed by the disease detection apparatus of this invention is of amicroscopic nature and can be a single cell, a single biologicalmolecular (e.g., DNA, RNA, or protein), a single biological subject(e.g., a single cell or virus), and other sufficiently small unit orfundamental biological composition.

As used herein, a “micro-device” or “micro device” can be any of a widerange of materials, properties, shapes, and degree of complexity andintegration. The term has a general meaning for an application from asingle material to a very complex device comprising multiple materialswith multiple sub units and multiple functions. The complexitycontemplated in the present invention ranges from a very small, singleparticle with a set of desired properties to a fairly complicated,integrated unit with various functional units contained therein. Forexample, a simple micro-device could be a single spherical article ofmanufacture of a diameter as small as 100 angstroms with a desiredhardness, a desired surface charge, or a desired organic chemistryabsorbed on its surface. A more complex micro device could be a 1millimeter device with a sensor, a simple calculator, a memory unit, alogic unit, and a cutter all integrated onto it. In the former case, theparticle can be formed via a fumed or colloidal precipitation process,while the device with various components integrated onto it can befabricated using various integrated circuit manufacturing processes.

A micro device used in the present invention can range in size (e.g.,diameter) from on the order of about 1 angstrom to on the order of about5 millimeters. For instance, a micro-device ranging in size from on theorder of about 10 angstroms to on the order of 100 microns can be usedin this invention for targeting biological molecules, entities orcompositions of small sizes such as cell structures, DNA, and bacteria.Or, a micro-device ranging in size from on the order of about one micronto the order of about 5 millimeters can be used in the present inventionfor targeting relatively large biological matters such as a portion of ahuman organ. As an example, a simple micro device defined in the presentapplication can be a single particle of a diameter less than 100angstroms, with desired surface properties (e.g., with surface charge ora chemical coating) for preferential absorption or adsorption into atargeted type of cell.

The present invention further provides an apparatus for detecting adisease in a biological subject, which comprises a pre-processing unit,a probing and detecting unit, a signal processing unit, and a disposalprocessing unit.

In some embodiments of the apparatus, the pre-processing unit includes asample filtration unit, a recharging unit, a constant pressure deliveryunit, and a sample pre-probing disturbing unit. This increases thecontraction ratio of certain substance of interests (such as cancercells) and therefore makes the apparatus more effective and efficient indetecting the targeted biological subject (such as cancer cells).

In some embodiments, the filtration unit can filter off unwantedsubstance by physical filtration (e.g., based on the electronic chargeor size of the substance) or separation by chemical reaction (therebycompletely removing the undesirable substances), biochemical reaction,electro-mechanical reaction, electro-chemical reaction, or biologicalreaction.

In some embodiments, the sample filtration unit can include an entrancechannel, a disturbing fluid channel, an accelerating chamber, and aslit. The slit and the interior walls of the entrance channel define twochannels (e.g., a top channel and a bottom channel) wherein thebiological subject can be separated due to the differences in itsproperty (e.g., electrical or physical property).

In some embodiments, a bio-compatible fluid can be injected into thedisturbing fluid channel to separate the biological subject. Forexample, the bio-compatible fluid can be injected from the entrance ofthe disturbing fluid channel and deliver to an opening in the entrancechannel wall. The bio-compatible fluid can be liquid or semi-liquid, andcan include saline, water, plasma, an oxygen-rich liquid, or anycombination thereof.

In some other embodiments, the angle between the entrance channel andthe disturbing fluid channel ranges from about 0° to about 180° (e.g.,from about 30° to about 150°, from about 60° to about 120°, or fromabout 75° to about 105°, or about 90°).

In some other embodiments, the width of each channel can range fromabout 1 nm to about 1 mm (e.g., from about 2 nm to about 0.6 mm or fromabout 10 nm to about 0.2 mm).

In some other embodiments, at least one of the channels comprises oneprobing device attached to the channel's sidewall, and the probingdevice is capable of measuring at the microscopic level an electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical, electro-optical,electro-thermal, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-optical, bio-thermal, bio-physical,bio-electro-mechanical, bio-electro-chemical, bio-electro-optical,bio-electro-thermal, bio-mechanical-optical, bio-mechanical thermal,bio-thermal-optical, bio-electro-chemical-optical,bio-electro-mechanical optical, bio-electro-thermal-optical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject.

In some embodiments, at least one of the channels comprises at least twoprobing devices attached to the channel's sidewalls, and the probingdevices are capable of measuring at the microscopic level an electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical, electro-optical,electro-thermal, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-optical, bio-thermal, bio-physical,bio-electro-mechanical, bio-electro-chemical, bio-electro-optical,bio-electro-thermal, bio-mechanical-optical, bio-mechanical thermal,bio-thermal-optical, bio-electro-chemical-optical,bio-electro-mechanical optical, bio-electro-thermal-optical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject. The probing devicesmeasure the same or different properties at the same time or differenttimes.

The two or more probing devices can be placed with a desired distancebetween each other (at least 10 angstroms). Examples of the desireddistance include from about 5 nm to about 100 mm, from about 10 nm toabout 10 mm, from about 10 nm to about 5 mm, from about 10 nm to about 1mm, from about 15 nm to about 500 nm.

In some embodiments, the micro-device of this invention comprises atleast one probe and at least one detector. The probe can be utilized tolaunch a probing (disturbing or simulating) signal to probe (i.e.,disturb or stimulate) the biological subject, and the detector candetect the biological subject's response (signal) to the probing signal.As an example, a micro-device with at least one acoustic probe (such asan acoustic transducer or microphone) and at least one detector (such asan acoustic signal receiver) is utilized for biological subjectdetection, wherein the acoustic probe and detector may be constructedwith, among others, one or more piezo-electrical materials. In thisexample, an acoustic signal is first launched, and scanned across itsfrequency range (e.g., from sub Hz to over MHz) by the probe. Theresponse signal to the launched acoustic signal by the probe is thencollected by the detector, and subsequently recorded, amplified (e.g.,by a lock-in amplifier), and analyzed. The response signal containscharacteristic information of a biological subject that is tested. Forexample, depending on certain properties of the tested biologicalsubject, the detected acoustic resonant frequency, intensity, frequencyversus intensity spectrum, or intensity distribution by the detector mayindicate characteristic information about the tested biological subject.Such information includes density, density distribution, absorptionproperties, shape, surface properties, and other static and dynamicproperties of the biological subject.

In some embodiments, the sample filtration unit can include an entrancechannel, a biocompatible filter, an exit channel, or any combinationthereof. When a biological subject passes through the entrance channeltoward the exit channel, the biological subject of a size larger thanthe filter hole will be blocked against the exit channel, resulting inthe smaller biological subject being flushed out through the exitchannel. A biocompatible fluid is injected from the exit to carry thebiological subject accumulated around the filter and flush out from thechannel. The biological subject with a large size is then filtered forfurther analysis and detection in the detecting component or unit of theapparatus.

In some embodiments, the sample pre-probing disturbing unit can includeone micro-device with a channel, a slit located inside the channel, andoptionally two plates outside the channel. The two plates can apply asignal, e.g., an electronic voltage, to the biological subject travelingthrough the channel and separates it based on the electronic charge thebiological subject carries. The slit and the interior channels of thechannel define two channels where the separated biological subjectsenter and optionally are detected for its property at the microscopiclevel.

In some embodiments, the sample pre-probing disturbing unit applies tothe biological subject an electrical, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-optical, electro-thermal,electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-optical,bio-thermal, bio-physical, bio-electro-mechanical, bio-electro-chemical,bio-electro-optical, bio-electro-thermal, bio-mechanical-optical,bio-mechanical thermal, bio-thermal-optical,bio-electro-chemical-optical, bio-electro-mechanical optical,bio-electro-thermal-optical, bio-electro-chemical-mechanical, physicalor mechanical signal, or a combination thereof. The signal can beapplied, e.g., with the two plates described above or in other means(depending on the nature of the signal). The signal as applied can bepulsed or constant.

In some embodiments, the recharging unit recharges nutrient or respiringgas (such as oxygen) to the biological subject. Alternatively, it canalso clean up the metabolite of the biological subject. With such arecharging unit, the life stability of the biological subject in thesample is sustained and its use is extended, thereby giving moreaccurate and reliable detecting results. Examples of nutrient includebiocompatible strong or weak electrolyte, amino acid, mineral, ions,oxygen, oxygen-rich liquid, intravenous drip, glucose, and protein.Another example of the nutrient is a solution containing nano-particlesthat can be selectively absorbed by certain biological subjects (e.g.,cells or viruses).

The recharging system can be separate from and outside of the othercomponents of the apparatus. Alternatively, it can also be installedwithin one of the other components, e.g., the probing and detecting unitor the disposal processing unit.

In some other embodiments, the signal processing unit comprises anamplifier (e.g., a lock-in amplifier), an A/D (alternate/directelectrical current or analog to digital) converter, a micro-computer, amanipulator, a display, and network connections.

In some instance, the signal processing unit collects more than onesignal (i.e., multiple signals), and the multiple signals can beintegrated to cancel out noise or to enhance the signal to noise ratio.The multiple signals can be signals from multiple locations or frommultiple times.

The invention further provides a method for detecting a disease withenhanced sensibility in a subject in need thereof, which comprises:taking a biological sample from the subject and taking a biologicalsample from a disease-free subject; optionally placing the biologicalsample in a biocompatible media; analyzing the two biological samples tomeasure a property thereof at the microscopic level with a micro-devicewhich comprises a first micro sensor for detecting a property of thebiological samples at the microscopic level, and an interior walldefining a channel, wherein the micro sensor is located in the interiorwall of the micro-device and detects the property of the biologicalsamples at the microscopic level, and the biological sample istransported within the channel; and comparing the measured property ofthe two biological samples.

In some embodiments, the micro-device further comprises a second microsensor for applying a probing signal on the biological samples or on theoptional media, thereby changing and optimizing (enhancing) the natureor value of the property to be detected at the microscopic level. Thisprocess would result in amplified or enhanced value of the property tobe detected, which in turn makes the property easier to detect andmeasure, thus increasing the sensibility of the detection and measure.The probing signal and the property to be detected can be of the sametype or different types. For example, the probing signal and theproperty to be detected can both be an electrical property or an opticalproperty or a mechanical property or a thermal property. Or, the probingsignal and the property to be detected can be, e.g., an optical propertyand an electrical property, an optical property and a magnetic property,an electrical property and a mechanic property, a mechanical propertyand an electrical property, a chemical property and a biologicalproperty, a physical property and an electrical property, an electricalproperty and a thermal property, a bio-chemical property and a physicalproperty, a bio-electro-mechanical property and a thermal property, abio-chemical property and an electrical property, a bio-chemicalproperty and an optical property, a bio-chemical property and a thermalproperty, a bio-chemical property and a chemical property, a biologicalproperty and an electrical property, a biological property and anoptical property, and a biological property and a thermal property,respectively.

Each of the probing signal and the property to be detected can be anelectrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-optical, electro-thermal, electro-chemical-mechanical,bio-chemical, bio-mechanical, bio-optical, bio-thermal, bio-physical,bio-electro-mechanical, bio-electro-chemical, bio-electro-optical,bio-electro-thermal, bio-mechanical-optical, bio-mechanical thermal,bio-thermal-optical, bio-electro-chemical-optical,bio-electro-mechanical optical, bio-electro-thermal-optical,bio-electro-chemical-mechanical, physical or mechanical property of thebiological subject, or a combination thereof.

In some embodiments, the change of the property is from a static stateto a dynamic or pulse state, or from a lower value to a higher value.

In some other embodiments, the probing signal or at least one of theparameters of the environmental setting in which the biological subjectto be measured resides is changed from one value to a new value, or froma static state to a dynamic state, in order to further enhance theproperty to be detected and thus optimize the measure sensibility of themicro-device. Such parameters or probing signal include, but are notlimited to, electrical, electro-magnetic, optical, thermal,bio-chemical, chemical, mechanical, physical, acoustical,bio-electrical, bio-optical, electro-optical, or a combination thereof.Specifically, examples of the probing signal and a property of the mediainclude, but are not limited to, laser intensity, temperature, catalystconcentration, acoustic energy, bio-marker concentration, electricalvoltage, electrical current, fluorescent dye concentration, the amountof agitation in the biological samples, and fluid flow rate.

Specifically, in order to enhance measurement sensitivity and maximizethe difference in signals between normal biological samples and diseasedbiological samples, applied probing (disturbing) signal and/or at leastone of the parameters of the environmental surrounding in which thebiological sample resides is intentionally changed from one value to anew value, or from a static value (DC value) to a pulsed value (ACvalue). The new value can be optimized to trigger maximum response fromthe biological sample. The new value can also be optimized to obtainenhanced difference in measured signals between the normal biologicalsample and diseased sample, resulting in enhanced measurementsensitivity. For example, for making dynamic measurements to furtherenhance measurement sensitivity, during measurements, at least one ofthe parameters applied to the biological sample being measured or atleast one of the properties in the surrounding media (in which thebiological sample resides) is intentionally changed from a static state(constant value) to a dynamic state (for example, a pulsed value or analternating value), or from one value to a new value. As a novelexample, in a measurement, a DC current applied to a biological sampleis intentionally changed to an AC current. In another novel example, aconstant temperature applied to a biological sample is changed to ahigher temperature, or a pulsed heat wave (for example, from 30° C. to50° C., then from 50° C. back to 30° C.). The above disclosed inventivemethod (the utilization of dynamic probing (disturbing or stimulating)signal, optimized probing (disturbing or stimulating) value and probingsignal ramp-up speed) can also be used in conjunction with variouslock-in techniques including but not limited to phase lock-in techniqueand/or the use of pulsed or alternating probing signal with signalamplification synchronized to the frequency of the probing signal.

Biological subjects that can be detected by the apparatus include, e.g.,blood, urine, saliva, tear, and sweat. The detection results canindicate the possible occurrence or presence of a disease (e.g., one inits early stage) in the biological subject.

As used herein, the term “absorption” typically means a physical bondingbetween the surface and the material attached to it (absorbed onto it,in this case). On the other hand, the word “adsorption” generally meansa stronger, chemical bonding between the two. These properties are veryimportant for the present invention as they can be effectively used fortargeted attachment by desired micro devices for measurement at themicroscopic level.

As used herein, the term “contact” (as in “the first micro-devicecontacts a biologic entity”) is meant to include both “direct” (orphysical) contact and “non-direct” (or indirect or non-physical)contact. When two subjects are in “direct” contact, there is generallyno measurable space or distance between the contact points of these twosubjects; whereas when they are in “indirect” contact, there is ameasurable space or distance between the contact points of these twosubjects.

As used herein, the term “probe” or “probing,” in addition to itsdictionary meaning, could mean applying a signal (e.g., an electrical,acoustic, magnetic or thermal signal) to a subject and therebystimulating the subject and causing it to have some kind of intrinsicresponse.

As used herein, the term “electrical property” refers to surface charge,surface potential, electrical field, charge distribution, electricalfield distribution, resting potential, action potential, or impedance ofa biological subject to be analyzed.

As used herein, the term “magnetic property” refers to diamagnetic,paramagnetic, or ferromagnetic.

As used herein, the term “electromagnetic property” refers to propertythat has both electrical and magnetic dimensions.

As used herein, the term “thermal property” refers to temperature,freezing point, melting point, evaporation temperature, glass transitiontemperature, or thermal conductivity.

As used herein, the term “optical property” refers to reflection,optical absorption, optical scattering, wave length dependentproperties, color, luster, brilliance, scintillation, or dispersion.

As used herein, the term “acoustical property” refers to thecharacteristics found within a structure that determine the quality ofsound in its relevance to hearing. It can generally be measured by theacoustic absorption coefficient. See, e.g., U.S. Pat. No. 3,915,016, formeans and methods for determining an acoustical property of a material;T. J. Cox et al., Acoustic Absorbers and Diffusers, 2004, Spon Press.

As used herein, the term “biological property” is meant to generallyinclude chemical and physical properties of a biological subject.

As used herein, the term “chemical property” refers to pH value, ionicstrength, or bonding strength within the biological sample.

As used herein, the term “physical property” refers to any measurableproperty the value of which describes a physical system's state at anygiven moment in time. The physical properties of a biological sample mayinclude, but are not limited to absorption, albedo, area, brittleness,boiling point, capacitance, color, concentration, density, dielectrical,electrical charge, electrical conductivity, electrical impedance,electrical field, electrical potential, emission, flow rate, fluidity,frequency, inductance, intrinsic impedance, intensity, irradiance,luminance, luster, malleability, magnetic field, magnetic flux, mass,melting point, momentum, permeability, permittivity, pressure, radiance,solubility, specific heat, strength, temperature, tension, thermalconductivity, flow rate, velocity, viscosity, volume, surface area,shape, and wave impedance.

As used herein, the term “mechanical property” refers to strength,hardness, flow rate, viscosity, toughness, elasticity, plasticity,brittleness, ductility, shear strength, elongation strength, fracturestress, or adhesion of the biological sample.

As used herein, the term “disturbing signal” has the same meaning as“probing signal” and “stimulating signal.”

As used herein, the term “disturbing unit” has the same meaning as“probing unit” and “stimulating unit.”

As used herein, the term “conductive material” (or its equivalent“electrical conductor”) is a material which contains movable electricalcharges. A conductive material can be a metal (e.g., copper, silver, orgold) or non-metallic (e.g., graphite, solutions of salts, plasmas, orconductive polymers). In metallic conductors, such as copper oraluminum, the movable charged particles are electrons (see electricalconduction). Positive charges may also be mobile in the form of atoms ina lattice that are missing electrons (known as holes), or in the form ofions, such as in the electrolyte of a battery.

As used herein, the term “electrically insulating material” (also knownas “insulator” or “dielectric”) refers to a material that resists theflow of electrical current. An insulating material has atoms withtightly bonded valence electrons. Examples of electrically insulatingmaterials include glass or organic polymers (e.g., rubber, plastics, orTeflon).

As used herein, the term “semiconductor” (also known as “semiconductingmaterial”) refers to a material with electrical conductivity due toelectron flow (as opposed to ionic conductivity) intermediate inmagnitude between that of a conductor and an insulator. Examples ofinorganic semiconductors include silicon, silicon-based materials, andgermanium. Examples of organic semiconductors include such aromatichydrocarbons as the polycyclic aromatic compounds pentacene, anthracene,and rubrene; and polymeric organic semiconductors such aspoly(3-hexylthiophene), poly(p-phenylene vinylene), polyacetylene andits derivatives. Semiconducting materials can be crystalline solids(e.g., silicon), amorphous (e.g., hydrogenated amorphous silicon andmixtures of arsenic, selenium and tellurium in a variety ofproportions), or even liquid.

As used herein, the term “biological material” has the same meaning of“biomaterial” as understood by a person skilled in the art. Withoutlimiting its meaning, biological materials or biomaterials can generallybe produced either in nature or synthesized in the laboratory using avariety of chemical approaches utilizing organic compounds (e.g., smallorganic molecules or polymers) or inorganic compounds (e.g., metalliccomponents or ceramics). They generally can be used or adapted for amedical application, and thus comprise whole or part of a livingstructure or biomedical device which performs, augments, or replaces anatural function. Such functions may be benign, like being used for aheart valve, or may be bioactive with a more interactive functionalitysuch as hydroxyl-apatite coated hip implants. Biomaterials can also beused every day in dental applications, surgery, and drug delivery. Forinstance, a construct with impregnated pharmaceutical products can beplaced into the body, which permits the prolonged release of a drug overan extended period of time. A biomaterial may also be an autograft,allograft, or xenograft which can be used as a transplant material. Allthese materials that have found applications in other medical orbiomedical fields can also be used in the present invention.

As used herein, the term “microelectronic technology or process”generally encompasses the technologies or processes used for fabricatingmicro-electronic and optical-electronic components. Examples includelithography, etching (e.g., wet etching, dry etching, or vapor etching),oxidation, diffusion, implantation, annealing, film deposition,cleaning, direct-writing, polishing, planarization (e.g., by chemicalmechanical polishing), epitaxial growth, metallization, processintegration, simulation, or any combinations thereof. Additionaldescriptions on microelectronic technologies or processes can be foundin, e.g., Jaeger, Introduction to Microelectronic Fabrication, 2^(nd)Ed., Prentice Hall, 2002; Ralph E. Williams, Modern GaAs ProcessingMethods, 2^(nd) Ed., Artech House, 1990; Robert F. Pierret, AdvancedSemiconductor Fundamentals, 2^(nd) Ed., Prentice Hall, 2002; S.Campbell, The Science and Engineering of Microelectronic Fabrication,2^(nd) Ed., Oxford University Press, 2001, the contents of all of whichare incorporated herein by reference in their entireties.

As used herein, the term “selective” as included in, e.g., “patterningmaterial B using a microelectronics process selective to material A”,means that the microelectronics process is effective on material B butnot on material A, or is substantially more effective on material B thanon material B (e.g., resulting in a much higher removal rate on materialB than on material A and thus removing much more material B thanmaterial A).

As used herein, the term “carbon nano-tube” generally refers to asallotropes of carbon with a cylindrical nanostructure. See, e.g., CarbonNanotube Science, by P. J. F. Harris, Cambridge University Press, 2009,for more details about carbon nano-tubes.

Through the use of a single micro-device or a combination ofmicro-devices integrated into a disease detection apparatus, the diseasedetection capabilities can be significantly improved in terms ofsensitivity, specificity, speed, cost, apparatus size, functionality,and ease of use, along with reduced invasiveness and side-effects. Alarge number of micro-device types capable of measuring a wide range ofmicroscopic properties of biological sample for disease detection can beintegrated and fabricated into a single detection apparatus usingmicro-fabrication technologies and novel process flows disclosed herein.While for the purposes of demonstration and illustration, a few novel,detailed examples have been shown herein on how microelectronics ornano-fabrication techniques and associated process flows can be utilizedto fabricate highly sensitive, multi-functional, and miniaturizeddetection devices, the principle and general approaches of employingmicroelectronics and nano-fabrication technologies in the design andfabrication of high performance detection devices have been contemplatedand taught, which can and should be expanded to various combination offabrication processes including but not limited to thin film deposition,patterning (lithography and etch), planarization (including chemicalmechanical polishing), ion implantation, diffusion, cleaning, variousmaterials, and various process sequences and flows and combinationsthereof.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1A is a perspective illustration of a disease detection apparatusof this invention in which a biological sample placed in it or movingthrough it can be tested. FIG. 1B and FIG. 1C illustrate the apparatuswhich comprises multiple individual detection micro-devices.

FIG. 2A is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention with multiple micro-devices. FIGS.2B-2L are perspective illustrations of the novel process flow forfabricating the micro-device. FIGS. 2M-2N are cross-sectional views ofan apparatus comprising multiple individual micro-devices.

FIG. 3 is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention with multiple micro-devices ofdifferent detection probes.

FIG. 4 is a perspective illustration of a disease detection apparatus ofthis invention.

FIGS. 5A-5L illustrate a novel process flow for fabricating a diseasedetection apparatus of this invention utilizing microelectronicstechnologies.

FIGS. 6A-6B are perspective illustrations of a disease detectionapparatus fabricated by a method of this invention. The apparatus iscapable of probing a single cell and measuring its microscopicproperties.

FIG. 7 is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention with multiple micro-devices placedat a desired distance for time of flight measurements.

FIGS. 8A-8G are perspective illustrations of a novel set of microscopicprobes.

FIG. 9 is a perspective illustration of a novel four-point probe.

FIGS. 10A-10R illustrate a novel process flow for fabricating a class ofmicro-devices capable of trapping, sorting, probing, measuring, andmodifying a biological subject at the microscopic level and in athree-dimensional space.

FIGS. 11A-11L illustrate a novel process flow for fabricating a class ofmicro-devices capable of measuring physical properties of a biologicalsubject and other properties related to cell membrane.

FIGS. 12A-12B illustrate how a micro-device with two micro-probescapable of moving in opposite directions when a force is applied can beutilized to probe properties of a biological subject (e.g., mechanicalproperties of a cell membrane).

FIGS. 13A-13B illustrate a novel time of flight detection arrangementfor disease detection applications.

FIGS. 14A-14B illustrate yet another time of flight disease detectionarrangement.

FIGS. 15A-15F illustrate another novel time of flight disease detectionapplication.

FIG. 16 illustrates a fluid delivery system.

FIGS. 17B-17C illustrate a novel device which can engage in cellularcommunications at the single cell level. FIG. 17A illustrates how thesignal is processed and responded in a single cell.

FIG. 18 illustrates a system block diagram of a disease detectionapparatus, comprising various functional modules.

FIGS. 19A-19L illustrate a micro-device capable of communicating,trapping, sorting, analyzing, treating, or modifying a DNA and measuringthe DNA's various properties.

FIGS. 20A-20C illustrate an apparatus of this invention that can detectthe surface charge on biological subjects and separate them by a slitbased on the charge.

FIGS. 21A-21B illustrate another apparatus of this invention that candetect the optical properties of the biological subject with a set ofoptical sensors.

FIG. 22 illustrates another apparatus of this invention that canseparate biological subjects of different geometric size and detecttheir properties respectively.

FIG. 23 illustrates an apparatus of this invention that can measure theacoustic property of a biological subject.

FIG. 24 illustrates an apparatus of this invention that can measure theinternal pressure of a biological subject.

FIGS. 25A-25C illustrate an apparatus of this invention that hasconcaves between the probe couples, in the bottom or ceiling of thechannel.

FIGS. 26A-26B illustrate another apparatus of this invention that hasconcaves of a different shape from those illustrated in FIG. 25.

FIG. 27 illustrates an apparatus of this invention that has a steppedchannel.

FIG. 28 illustrates an apparatus of this invention that has a set ofthermal meters.

FIG. 29 illustrates an apparatus of this invention that includes acarbon nano-tube as the channel with DNA contained therein.

FIGS. 30A-30G illustrate an integrated apparatus of this invention thatincludes a detecting device and an optical sensor.

FIGS. 31A-31D illustrate an integrated apparatus of this invention thatincludes a detecting device and a logic circuitry.

FIGS. 32A-32C illustrate an apparatus of this invention that includes adetecting device and a filter.

FIG. 33 illustrates how micro-devices of this invention can be used tomeasure the geometric factors of DNA.

FIG. 34 illustrates a process for fabricating a micro-device of thisinvention with a cover atop the trench to form a channel.

FIG. 35 is a diagram of an apparatus of this invention for detecting adisease in a biological subject.

FIG. 36 shows an example of a sample filtration unit.

FIGS. 37A-37B show another example of a sample filtration unit.

FIG. 38 is a diagram of a pre-processing unit of an apparatus of thisinvention.

FIG. 39 is a diagram of an information processing unit of an apparatusof this invention.

FIG. 40 shows the integration of multiple signals which results incancellation of noise and enhancement of signal/noise ratio.

FIGS. 41A-41G show one embodiment of the fabrication process of thisinvention for manufacturing a detection device with at least onedetection chamber and at least one detector.

FIGS. 42A-42I show another embodiment of a process of this invention formanufacturing a detection device with enclosed detection chambers,detectors, and channels for transporting biological samples such asfluidic samples.

FIGS. 43A-43C show a novel disease detection method in which at leastone probe object is launched at a desired speed and direction toward abiological subject, resulting in a collision.

FIGS. 44A-44H illustrate a novel fabrication process of this inventionfor forming multiple components with different materials at the samedevice level.

FIG. 45 shows a process of this invention for detecting a biologicalsubject using a disease detection device.

FIGS. 46A-46B show another embodiment of disease detection processwherein diseased and healthy biological subjects are separated and thediseased biological subjects are delivered to further test.

FIGS. 47A-47B are an arrayed biological detecting device wherein aseries of detecting devices are fabricated into an apparatus.

FIGS. 48A-48B show another embodiment of a disease detection device ofthe current invention including inlet and outlet of the device, thechannel where the biological subject passes through, and detectiondevices aligned along the walls of the channel.

FIGS. 49A-49I show a schedule for fabricating a piezo-electricalmicro-detector of this invention.

FIGS. 50A-50F show an example of the micro-device of this inventionpackaged and ready for use.

FIGS. 51A-51F show another example of the micro-device of this inventionthat is packaged and ready for use.

FIGS. 52A-52D show yet another example of the micro-device of thisinvention that is packaged and ready for use.

FIGS. 53A-53C show a micro-device of this invention that has a channel(trench) and an array of micro sensors.

FIGS. 54A-54E show another micro-device of this invention that comprises2 panels one of which has an array of micro sensors and two microcylinders.

FIGS. 55A-55C show a micro-device of this invention that comprises 2panels one of which has an array of micro sensors and two microcylinders both of which have a probing sensor.

FIGS. 56A-56B show another micro-device of this invention comprisingseveral “sub-devices.”

FIG. 57 shows an example of the micro-devices of this invention whichincludes an application specific integrated circuit (ASIC) chip with I/Opads.

FIGS. 58A-58G are a diagram of the underlying principal of themicro-device of this invention which functions by combining variouspre-screening and detection methods in unobvious ways.

FIGS. 59A-59C show cross-sectional and outside views of a channel intowhich a biological subject can flow.

FIGS. 60A-60E show a biological subject to be detected passing through achannel aligned with detectors along its passage in a micro-device ofthis invention.

FIGS. 61A-61N illustrate a device fabrication process flow andassociated device structures.

FIGS. 62A-62B are views of the micro-device of this invention showingone or two sorting units therein.

FIG. 63 shows a micro-device of this invention with a high number ofdesired structures fabricated simultaneously on the same chip.

FIG. 64 shows another micro-device of this invention for sorting,screening, separating, probing and detecting diseased biologicalentities.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to apparatus for detectingdisease in a biological subject in vivo or in vitro (e.g., human being,an organ, a tissue, or cells in a culture). Each apparatus includes abiological fluid delivering system and a probing and detecting device.The apparatus is capable of measuring microscopic properties of abiological sample. By the constant pressure fluid delivery system,microscopic biological subjects can be delivered onto or into thediagnostic micro-device of the apparatus. Compared to traditionaldetection apparatus or technologies, the apparatus provided by thisinvention are advantageous in providing enhanced detection sensitivity,specificity, and speed, with reduced costs and size. The apparatus canfurther include a biological interface, a probing controlling and dataanalysis circuitry, or a system reclaiming or treating medical waste.Additional micro-devices, e.g., a second detection device, can also beincluded or integrated into the apparatus for enhanced detectioncapabilities.

As a key component of the apparatus, the micro-device should includemeans to perform at least the function of addressing, controlling,forcing, receiving, amplifying, or storing information from each probingaddress. As an example, such means can be a central control unit thatincludes a controlling circuitry, an addressing unit, an amplifiercircuitry, a logic processing circuitry, a memory unit, an applicationspecific chip, a signal transmitter, a signal receiver, or a sensor.

In some embodiments, the fluid delivering system comprises a pressuregenerator, a pressure regulator, a throttle valve, a pressure gauge, anddistributing kits. As examples of these embodiments, the pressuregenerator can include a motor piston system and a bin containingcompressed gas; the pressure regulator (which can consist of multipleregulators) can down-regulate or up-regulate the pressure to a desiredvalue; the pressure gauge feeds back the measured value to the throttlevalve which then regulates the pressure to approach the target value.

The biological fluid to be delivered can be a sample of a biologicalsubject to be detected for disease or something not necessarily to bedetected for disease. In some embodiment, the fluid to be delivered isliquid (e.g., a blood sample, a urine sample, or a saline) or gas (e.g.,nitrogen, argon, helium, neon, krypton, xenon, or radon). The pressureregulator can be a single pressure regulator or multiple pressureregulators which are placed in succession to either down-regulate orup-regulate the pressure to a desired level, particularly when theinitial pressure is either too high or too low for a single regulator toadjust to the desired level or a level that is acceptable for an enddevice or target.

In some other embodiments, the system controller includes apre-amplifier, a lock-in amplifier, an electrical meter, a thermalmeter, a switching matrix, a system bus, a nonvolatile storage device, arandom access memory, a processor, or a user interface. The interfacecan include a sensor which can be a thermal sensor, a flow meter, apiezo-meter, or another sensor.

In still some other embodiments, apparatus of this invention furtherinclude a biological interface, a system controller, a system forreclaiming or treatment medical waste. The reclaiming and treatment ofmedical waste can be performed by the same system or two differentsystems.

Another aspect of this invention provides apparatus for interacting witha cell, which include a device for sending a signal to the cell andoptionally receiving a response to the signal from the cell.

In some embodiments, the interaction with the cell can be probing,detecting, communicating with, treating, or modifying with a codedsignal that can be an electrical, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical signal, or acombination thereof.

In some other embodiments, the device contained in the apparatus caninclude multiple surfaces coated with one or more elements orcombinations of elements, and a control system for releasing theelements. In some instances, the control system can cause release of theelements from the device surface via thermal energy, optical energy,acoustic energy, electrical energy, electro-magnetic energy, magneticenergy, radiation energy, or mechanical energy in a controlled manner.The energy can be in the pulsed form at desired frequencies.

In some other embodiments, the device contained in the apparatus includea first component for storing or releasing one element or a combinationof elements onto the surface of the cell or into the cell; and a secondcomponent for controlling the release of the elements (e.g., a circuitryfor controlling the release of the elements). The elements can be abiological component, a chemical compound, Ca, C, Cl, Co, Cu, H, I, Fe,Mg, Mn, N, O, P, F, K, Na, S, Zn, or a combination thereof. The signal,pulsed or constant, can be in the form of a released element orcombination of elements, and it can be carried in a liquid solution,gas, or a combination thereof. In some instances, the signal can be at afrequency ranging from about 1×10⁻⁴ Hz to about 100 MHz or ranging fromabout 1×10⁻⁴ Hz to about 10 Hz, or at an oscillation concentrationranging from about 1.0 nmol/L to about 10.0 mmol/L. Also, the signalcomprises the oscillation of a biological component, a chemicalcompound, Ca, C, Cl, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, Zn,or a combination thereof, e.g., at desired oscillating frequencies.

In some embodiments, the signal to be sent to the cell can be in theform of oscillating element, compound, or an oscillating density of abiological component, and a response to the signal from the cell is inthe form of oscillating element, compound, or an oscillating density ofa biological component.

In some embodiments, the device can be coated with a biological film,e.g., to enhance compatibility between the device and the cell.

In some other embodiments, the device can include components forgenerating a signal to be sent to the cell, receiving a response to thesignal from the cell, analyzing the response, processing the response,and interfacing between the device and the cell.

Still another aspect of this invention provides devices each including amicro-filter, a shutter, a cell counter, a selector, a micro-surgicalkit, a timer, and a data processing circuitry. The micro-filter candiscriminate abnormal cells by a physical property (e.g., e.g.,dimension, shape, or velocity), mechanical property, electricalproperty, magnetic property, electromagnetic, thermal property (e.g.,temperature), optical property, acoustical property, biologicalproperty, chemical property, or bio-chemical property. The devices eachcan also include one or more micro-filters. Each of these micro-filterscan be integrated with two cell counters, one of which is installed atthe entrance of each filter well, while the other is installed at theexit of each filter well. The shape of the micro-filter's well isrectangle, ellipse, circle, or polygon; and the micro-filter's dimensionranges from about 0.1 μm to about 500 μm or from about 5 um to about 200um. As used herein, the term “dimension” means the physical or featuresize of the filter opening, e.g., diameter, length, width, or height.The filter can be coated with a biological or bio-compatible film, e.g.,to enhance compatibility between the device and the cell.

In some embodiments of these devices, the shutter sandwiched by twofilter membranes can be controlled by a timer (thus time shutter). Thetimer can be triggered by the cell counter. For instance, when a cellpasses through the cell counter of the filter entrance, the clock istriggered to reset the shutter to default position, and moves at apreset speed towards the cell pathway, and the timer records the time asthe cell pass through the cell counter at the exit.

Still a further aspect of this invention provides methods forfabricating a micro-device with micro-trench and probe embedded in themicro-trench's sidewalls. A micro-trench is an unclosed tunnel (see,e.g., FIG. 2I, 2030), which can be coupled with another upendedsymmetric trench (see, e.g., FIG. 2K, 2031) to form a closed channel(see, e.g., FIG. 2L, 2020). The method may include chemical vapordeposition, physical vapor deposition, or atomic layer deposition todeposit various materials on a substrate; lithography or etch totransfer patterns from design to structure; chemical mechanicalplanarization for surface planarization, chemical cleaning for particleremoval, diffusion or ion implantation for doping elements into specificlayers; or thermal anneal to reduce the crystal defects and activatediffused ions. An example of such method includes: depositing a firstmaterial onto a substrate; depositing a second material onto the firstmaterial and patterning the second material by a microelectronic process(e.g., lithography or etch) to form a detecting tip; depositing a thirdmaterial on the second material and then patterning the second materialby a planarization process; depositing a fourth material on the thirdmaterial and patterning the fourth material first by a microelectronicprocess (e.g., lithography or etch) and then by a microelectronicprocess (e.g., another etch) in which the fourth material serves as ahardmask. A hardmask generally refers to a material (e.g., inorganicdielectrical or metallic compound) used in semiconductor processing asan etch mask in lieu of polymer or other organic “soft” materials.

In some embodiments, the method further includes coupling two devicesthat are thus fabricated and symmetric (i.e., a flipped mirror) to forma detecting device with channels. The entrance of each channel can beoptionally bell-mouthed, e.g., such that the size of channel's openingend (the entrance) is larger than the channel's body, thereby making iteasier for a cell to enter the channel. The shape of each channel'scross-section can be rectangle, ellipse, circle, or polygon. Themicro-trenches of the coupled two micro-devices can be aligned by themodule of alignment marks designed on the layout of the micro-device.The dimension of the micro-trench can range from about 0.1 um to about500 um.

Alternatively, the method can also include covering the micro-trench ofthe micro-device with a flat panel. Such a panel can comprise or be madewith silicon, SiGe, SiO₂, Al₂O₃, or other optical materials. Examples ofother potentially suitable optical materials include acrylate polymer,AgInSbTe, synthetic alexandrite, arsenic triselenide, arsenictrisulfide, barium fluoride, CR-39, cadmium selenide, caesium cadmiumchloride, calcite, calcium fluoride, chalcogenide glass, galliumphosphide, GeSbTe, germanium, germanium dioxide, glass code, hydrogensilsesquioxane, Iceland spar, liquid crystal, lithium fluoride,lumicera, METATOY, magnesium fluoride, magnesium oxide, negative indexmetamaterials, neutron super mirror, phosphor, picarin, poly(methylmethacrylate), polycarbonate, potassium bromide, sapphire, scotophor,spectralon, speculum metal, split-ring resonator, strontium fluoride,yttrium aluminum garnet, yttrium lithium fluoride, yttriumorthovanadate, ZBLAN, zinc selenide, and zinc sulfide.

In other embodiments, the method can further include integrating threeor more micro-devices thus fabricated to yield an enhanced device withan array of the channels.

Yet still another aspect of this invention relates to micro-devices eachincluding a micro-trench, a probe embedded aside the trench's side wallsor bottom floor, a supporting structure to move the probe, and acontrolling circuitry, wherein the micro-device is capable of trapping,sorting, or modifying a DNA and measuring its properties (e.g.,electrical, thermal, or optical properties). The micro-trench can beutilized to encase the DNA double helix.

In some embodiments, the width of the micro-trench ranges from about 1nm to about 10 μm, the depth of the micro-trench ranges from about 1 nmto about 10 μm, or the length of the micro-trench ranges from about 1 nmto about 10 mm. The probe can include or be made of a conductivematerial and, optionally, a flexible supporting structure to extend orcontract the probe. The probe can also have a tip aside the trench andthe tip matches spatially with either a major groove or a minor grooveof the DNA. The tip can match spatially with interlaced grooves of theDNA, which can be variable. The tip of can also match the end of eachstrand of the DNA helix. In some examples, the tip's diameter can rangefrom about 1 angstrom to about 10 μm.

In some other embodiments, the micro-device can further include an arrayof trenches, e.g., to enhance the efficiency.

Another aspect of this invention relates to a set of novel process flowsfor fabricating micro-devices (including micro-probes andmicro-indentation probes) for their applications in disease detection bymeasuring microscopic properties of a biological sample. Themicro-devices can be integrated into a disease detection apparatus ofthis invention to measure one or more properties at microscopic levels.

Another aspect of this invention is to involve in cellularcommunications and regulate cellular decision or response (such asdifferentiation, dedifferentiation, cell division and cell death) withfabricated signals. This could be further employed to detect and treatdiseases.

To further enhance measurement capabilities, multiple micro-devices canbe implemented into a piece of detection apparatus employing the time offlight technique, in which at least one probing micro-device and onesensing micro-device placed at a preset, known distance. The probingmicro-device can apply a signal (e.g., a voltage, a charge, anelectrical field, a laser beam, or an acoustic wave) to the biologicalsample to be measured, and the detection (sensing) micro-device canmeasure response from or of the biological sample after the sample hastraveled a known distance and a desired period of time. For instance, aprobing micro-device can apply an electrical charge to a cell first, andthen a detection (sensing) micro-device subsequently measures thesurface charge after a desired period of time (T) has lapsed and thecell has traveled a certain distance (L).

The micro-devices contained in the apparatus of this invention can havea wide range of designs, structures, functionalities, and applicationsdue to their diverse properties, high degree of flexibilities, andability of integration and miniaturization. They include, e.g., avoltage comparator, a four point probe, a calculator, a logic circuitry,a memory unit, a micro cutter, a micro hammer, a micro shield, a microdye, a micro pin, a micro knife, a micro needle, a micro thread holder,micro tweezers, a micro optical absorber, a micro mirror, a microwheeler, a micro filter, a micro chopper, a micro shredder, micro pumps,a micro absorber, a micro signal detector, a micro driller, a microsucker, a micro tester, a micro container, a signal transmitter, asignal generator, a friction sensor, an electrical charge sensor, atemperature sensor, a hardness detector, an acoustic wave generator, anoptical wave generator, a heat generator, a micro refrigerator and acharge generator.

Further, it should be noted that advancements in manufacturingtechnologies have now made fabrications of a wide range of micro-devicesand integration of various functions onto the same device highlyfeasible and cost effective. The typical human cell size is about 10microns. Using state-of-the-art integrated circuit fabricationtechniques, the minimum feature size defined on a micro-device can be assmall as 0.1 micron or below. Thus, it is ideal to utilize the disclosedmicro-devices for biological applications.

In terms of materials for the micro-devices, the general principle orconsideration is the material's compatibility with a biological subject.Since the time in which a micro-device is in contact with a biologicalsample (e.g., a cell; a biological molecule such as DNA, RNA, orprotein; or a tissue or organ sample) may vary, depending on itsintended application, a different material or a different combination ofmaterials may be used to make the micro-device. In some special cases,the materials may dissolve in a given pH in a controlled manner and thusmay be selected as an appropriate material. Other considerations includecost, simplicity, ease of use and practicality. With the significantadvancements in micro fabrication technologies such as integratedcircuit manufacturing technology, highly integrated devices with minimumfeature size as small as 0.1 micron can now be made cost-effectively andcommercially. One good example is the design and fabrication of microelectro mechanical devices (MEMS), which now are being used in a widevariety of applications in the integrated circuit industry.

Set forth below are several illustrations or examples of apparatus ofthis invention containing a class of innovative micro-devices that areintegrated into the disease detection apparatus of this invention, andof their fabrication process.

FIGS. 1A-1C are perspective illustrations of a disease detectionapparatus of this invention 111 in which a biological sample 211 such asa blood sample placed in it or moving through it is tested. In thisfigure, an example of disease detection apparatus 111 is in the form ofa cylinder, in which a biological sample 211 flowing through it (fromthe left side to the right side in the figure) can be tested for one ormore properties at the microscopic levels.

To enhance detection speed and sensitivity, a large number ofmicro-devices can be integrated into a single disease detectionapparatus of this invention, such as the apparatus illustrated in FIG.1B and FIG. 1C with the micro-devices spaced to measure a large numberof desired entities (such as cells, DNAs, RNAs, proteins, etc.) in thebiological sample. To achieve the above requirements, the detectionapparatus should be optimized with its surface area maximized to contactthe biological sample and with large number of micro-devices integratedon the maximized surface.

FIG. 2A is a perspective, cross-sectional illustration of a diseasedetection apparatus of this invention 122 with multiple identicalmicro-devices 311. A biological sample such as a blood sample 211 placedin it or moving through it can be tested for one or more properties atthe microscopic levels including, e.g., electrical properties (such assurface charge, surface potential, current, impedance, other electricalproperties), magnetic properties, electromagnetic properties, mechanicalproperties (such as density, hardness, shear strength, elongationstrength, fracture tress, and adhesion), biological features, chemicalproperties (e.g., pH or ionic strength), biochemical properties, thermalproperties (e.g., temperature), and optical properties.

Instead of measuring a single property of a biological subject fordisease diagnosis, various micro-devices can be integrated into adetection apparatus to detect multiple properties. FIG. 3 is aperspective, cross-sectional illustration of a disease detectionapparatus of this invention 133 with multiple micro-devices 311, 312,313, 314, and 315, of different detection probes in which a sample 211such as a blood sample placed in it or moving through it can be testedfor multiple properties including but not limited to electricalproperties (e.g., surface charge, surface potential, and impedance),magnetic properties, electromagnetic properties, mechanical properties(e.g., density, hardness and adhesion), thermal properties (e.g.,temperature), biological properties, chemical properties (e.g., pH),physical properties, acoustical properties, and optical properties.

FIGS. 2B-2N illustrate a process flow of this invention for fabricatingmicro-devices for trapping, sorting, probing, measuring, and modifyingbiological subjects (e.g., a single cell, a DNA or RNA molecule). First,a material 2002 (e.g., a non-conducting material) and another material2003 (e.g., a conducting material) are sequentially deposited on asubstrate 2001 (see FIG. 2B and FIG. 2C). The first material 2003 isthen subsequently patterned by the lithography and etch processes (seeFIG. 2D). Another material 2004 is then deposited (as shown in FIG. 2E)and planarized (as shown in FIG. 2F). Another layer of material 2005 isdeposited (as shown in FIG. 2G) and patterned as a hard mask (as shownin FIG. 2H), then followed by etch (as shown in FIG. 2J), which isstopped on the substrate 2001. FIG. 2I is a perspective illustration ofthe device, while FIG. 2J is a vertical illustration of the device.

As shown in FIG. 2K, the device 2080 and a mirrored or symmetric device2081 can be coupled together (as shown in FIG. 2L). As such, theapparatus having the pathway with probe embedded in the sidewall isfabricated.

As illustrated in FIG. 2M and FIG. 2N, a large number of detectionmicro-devices can be integrated together to enhance the detectionefficiency.

As illustrated herein, it is desirable to optimize the detectionapparatus design to maximize measurement surface area, since the greaterthe surface area, the greater number of micro-devices that can be placedon the detection apparatus to simultaneously measure the sample, therebyincreasing detection speed and also minimizing the amount of sampleneeded for the test. FIG. 4 is a perspective illustration of a diseasedetection apparatus of this invention 144. It includes two slabsseparated by a narrow spacing with a sample such as a blood sample to bemeasured placed between the slabs, with multiple micro-devices placed atthe inner surfaces of the slabs to measure one or more properties of thesample at microscopic levels.

Yet another aspect of this invention relates to a set of novelfabrication process flows for making micro-devices for disease detectionpurposes. FIGS. 5A-5L illustrate a novel process flow for fabricating adisease detection apparatus utilizing microelectronics technologies andprocesses. First, a material 412 is deposited on a substrate 411 (FIG.5A). It is then patterned by photolithography and etching processes(FIG. 5B). Following the deposition, material 413 is planarized usingchemical mechanical polishing as shown in FIG. 5D. Recessed areas, inthe form of hole pattern, are next formed in material 413 usingphotolithography and etch processes, as shown in FIG. 5E, followed bythe deposition of material 414 (FIG. 5F). Material 414 above the surfaceof material 413 is removed by chemical mechanical polishing (FIG. 5G),followed by deposition of material 415. Material 415 is next patternedusing photolithography and etching processes (FIG. 51). Material 414 isnext deposited and its excess material above its substrate 415 isremoved by chemical mechanical polishing (FIGS. 5J and 5K). Finally, alight etch or short chemical mechanical polishing to material 415 iscarried out to recess material 415, selective to material 414 (FIG. 5L),resulting in slight protruding of material 414. Material 412 can be apiezo-electrical material. When a voltage is applied to it in the rightdirection, it will expand and push up, resulting in upward motion inmiddle tip in material 414. Thus, a micro-device with two probes capableof measuring a range of properties (including mechanical and electricalproperties) of biological samples is fabricated, using the above novelfabrication process flow.

Detection apparatus integrated with micro-devices disclosed in thisapplication is fully capable of detecting pre-chosen properties on asingle cell, a single DNA, a single RNA, or an individual, small sizedbiological matter level. FIGS. 6A-6B are perspective illustrations of amicro-device 555 fabricated by a novel process flow disclosed in thispatent application (e.g., novel process flow illustrated in FIGS. 5A-5Labove) and how such a device is capable of probing a single cell 666 andmeasuring the cell for collecting intended parameters. FIG. 6Aillustrated a perspective, cross-section of a micro-device 555 with apair of micro probes 531 and 520, where micro probe 531 is in the formof a tip and micro probe 520 is in the form of a ring. Both of microprobes 531 and 520 can be conductive and they can serve as a pair ofprobes to measure electrical properties of a biological sample. Microprobe 531 is in contact with a base 518 which can be a piezo-electricalmaterial. When a voltage is applied to the base 518 made of apiezo-electrical material, the base 518 can expand and push micro probetip 531 upward, which can be useful in measuring various properties of abiological sample such as a single cell. In FIG. 6B, micro-device 555 isshown to measure a single cell 666, using probe tip 531 penetratingthrough cell membrane 611 and into the cell's inner space 622, whileprobe ring 520 making contact with cell membrane 611 at the outsidesurface of the membrane. This way, the micro-device 555 can make variousmeasurements on the cell, including its electrical properties (e.g.,electrical potential, current across the cell membrane, surface chargeon the membrane, and impedance), mechanical properties (e.g., hardnesswhen probe tip 531 is designed as a micro-indentation probe), thermalproperties (e.g., temperature), physical properties, and chemicalproperties (e.g., pH).

In another further aspect, the invention provides the design,integration, and fabrication process flow of micro-devices capable ofmaking highly sensitive and advanced measurements on very weak signalsin biological systems for disease detection under complicatedenvironment with very weak signal and relatively high noise background.Those novel capabilities using the class of micro-devices disclosed inthis invention for disease detection include but not limited to makingdynamic measurements, real time measurements (such as time of flightmeasurements, and combination of using probe signal and detectingresponse signal), phase lock-in technique to reduce background noise,and 4-point probe techniques to measure very weak signals, and uniqueand novel probes to measure various electronic, electromagnetic andmagnetic properties of biological samples at the single cell (e.g., atelomere of DNA or chromosome), single molecule (e.g., DNA, RNA, orprotein), single biological subject (e.g., virus) level.

For example, in a time of flight approach to obtain dynamic informationon the biological sample (e.g., a cell, a substructure of a cell, a DNA,a RNA, or a virus), a first micro-device is first used to send a signalto perturb the biological subject to be diagnosed, and then a secondmicro-device is employed to accurately measure the response from thebiological subject. In one embodiment, the first micro-device and thesecond micro-device are positioned with a desired or pre-determineddistance L apart, with a biological subject to be measured flowing fromthe first micro-device towards the second micro-device. When thebiological subject passes the first micro-device, the first micro-devicesends a signal to the passing biological subject, and then the secondmicro-device detects the response or retention of the perturbationsignal on the biological subject. From the distance between the twomicro-devices, time interval, the nature of perturbation by the firstmicro-device, and measured changes on the biological subject during thetime of flight, microscopic and dynamic properties of the biologicalsubject can be obtained. In another embodiment, a first micro-device isused to probe the biological subject by applying a signal (e.g., anelectronic charge) and the response from the biological subject isdetected by a second micro-device as a function of time.

To further increase detection sensitivity, a novel detection process fordisease detection is used, in which time of flight technique isemployed. FIG. 7 is a perspective, cross-sectional illustration ofdetection apparatus 155 with multiple micro-devices 321 and 331 placedat a desired distance 700 for time of flight measurements to attaindynamic information on biological sample 211 (e.g., a cell) withenhanced measurement sensitivity, specificity, and speed. In this timeof flight measurement, one or more properties of the biological sample211 are first measured when the sample 211 passes the first micro-device321. The same properties are then measured again when the sample 211passes the second micro-device 331 after it has traveled the distance700. The change in properties of sample 211 from at micro-device 321 toat micro-device 331 indicates how it reacts with its surroundingenvironment (e.g., a particular biological environment) during thatperiod. It may also reveal information and provide insight on how itsproperties evolve with time. Alternatively, in the arrangement shown inFIG. 7, micro-device 321 could be used first as a probe to apply a probesignal (e.g., an electrical charge) to sample 211 as the sample passesthe micro-device 321. Subsequently, the response of the sample to theprobe signal can be detected by micro-device 331 as the sample passes it(e.g., change in the electrical charge on the sample during the flight).Measurements on biological sample 211 can be done via contact ornon-contact measurements. In one embodiment, an array of micro-devicescan be deployed at a desired spacing to measure properties of thebiological subject over time.

The utilization of micro-devices (e.g., made by using the fabricationprocess flows of this invention) as discussed above and illustrated inFIG. 7 can be helpful for detecting a set of new, microscopic propertiesof a biological sample (e.g., a cell, a cell substructure, or abiological molecule such as DNA or RNA or protein) that have not beenconsidered in existing detection technologies. Such microscopicproperties can be electrical, magnetic, electromagnetic, thermal,optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical properties, ora combination thereof, of a biological sample that is a singlebiological subject (such as a cell, a cell substructure, a biologicalmolecule—e.g., DNA, RNA, or protein—or a sample of a tissue or organ).It is known that biological matters includes from basic bonding such asOH, CO, and CH bonding, to complex, three dimensional structures such asDNA and RNA. Some of them have a unique signature in terms of itselectronic configuration. Some of them may have unique electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical properties andconfigurations, or a combination thereof. Normal biological subject anddiseased biological subject may carry different signatures withrespective to the above said properties. However, none of the abovestated parameters or properties have been routinely used as a diseasedetection property. Using a disease detection apparatus including one ormore micro-devices of this invention, those properties can be detected,measured, and utilized as useful signals for disease detection,particularly for early stage detection of serious diseases such ascancer.

FIGS. 8A-8G are perspective illustrations of a novel set of microscopicprobes 341, 342, 343, 344, 345, 346, and 347 designed and configured todetect various electronic, magnetic, or electromagnetic states,configurations, or other properties at microscopic level on biologicalsamples 212, 213, 214, and 215, which can be a single cell, DNA, RNA,and tissue or sample. As an example, in terms of measuring electronicproperties, the shapes of biological samples 212, 213, 214, and 215 inFIGS. 8A-8G may represent electronic monopole (sample 212), dipole(samples 213 and 214), and quadruple (sample 215). The micro-devices341, 342, 343, 344, 345, 346, and 347 are optimized to maximizemeasurement sensitivity of those said parameters including but notlimited to electronic states, electronic charge, electronic clouddistribution, electrical field, and magnetic and electromagneticproperties, and the micro-devices can be designed and arranged in threedimensional configurations. For some diseases such as cancer, it islikely that electronic states and corresponding electronic propertiesdiffer between normal and cancerous cells, DNA, RNA, and tissue.Therefore, by measuring electronic, magnetic and electromagneticproperties at microscopic levels including at cell, DNA, and RNA levels,disease detection sensitivity and specificity can be improved.

In addition to the above examples in measuring electrical properties(e.g., charge, electronic states, electronic charge, electronic clouddistribution, electrical field, current, and electrical potential, andimpedance), mechanical properties (e.g., hardness, density, shearstrength, and fracture strength) and chemical properties (e.g., pH) in asingle cell, and in FIGS. 8A-8G for measuring electrical, magnetic orelectromagnetic states or configurations of biological samples at celland biological molecular (e.g., DNA, RNA, and protein) levels, othermicro-devices are disclosed in this application for sensitive electricalmeasurements.

FIG. 9 is a perspective illustration of a four-point probe for detectingweak electronic signal in a biological sample such as a cell, where afour point probe 348 is designed to measure electrical properties(impedance and weak electrical current) of a biological sample 216.

One of the key aspects of this invention is the design and fabricationprocess flows of micro-devices and methods of use the micro-devices forcatching and/or measuring biological subjects (e.g., cells, cellsubstructures, DNA, and RNA) at microscopic levels and in threedimensional space, in which the micro-devices have micro-probes arrangedin three dimensional manner with feature sizes as small as a cell, DNA,or RNA, and capable of trapping, sorting, probing, measuring, andmodifying biological subjects. Such micro-devices can be fabricatedusing state-of-the-art microelectronics processing techniques such asthose used in fabricating integrated circuits. Using thin filmdeposition technologies such as molecular epitaxy beam (MEB) and atomiclayer deposition (ALD), film thickness as thin as a few monolayers canbe achieved (e.g., 4 A to 10 A). Further, using electron beam or x-raylithography, device feature size on the order of nanometers can beobtained, making micro-device capable of trapping, probing, measuring,and modifying a biological subject (e.g., a single cell, a single DNA orRNA molecule) possible.

FIGS. 10A-10R illustrate a process flow of this invention forfabricating micro-devices for trapping, sorting, probing, measuring, andmodifying biological subjects (e.g., a single cell, a DNA or RNAmolecule). In this process flow, microelectronics processes are utilizedto fabricate micro-devices designed to achieve the above stated uniquefunctions. Specifically, a first material 712 (typically a conductingmaterial) is first deposited on a substrate 711 (FIG. 10A and FIG. 10B).The first material 712 is subsequently patterned by using lithographyand etch processes (FIG. 10C). A second material 713 is then depositedand planarized using chemical mechanical polishing process to removeoverburden of the second material 713 above the first material 712 (asshown in FIG. 10E). Another layer of material 714 is deposited andpatterned, followed by deposition and planarization by chemicalmechanical polishing of another layer of 712 (FIG. 10F). Next, a thirdmaterial 715 is deposited and patterned, using lithography and etchprocesses (FIG. 10G and FIG. 10H), followed by deposition andplanarization of a fourth material 716, typically a sacrificial material(FIG. 10I and FIG. 10J). Repeating the process flow of deposition ofpatterning material 712 or material 715 alternatively, and deposition ofmaterial 716 and planarization by chemical mechanical polishing (FIGS.10K-10M), a film stack featuring multiple layers with alternatingmaterial 712 (e.g., a conducting material) and material 715 (e.g., aninsulating material) in at least portions of the device is formed.Finally, material 716 between film stacks 771 and 772 is removed by wetetch, dry etch (which may require lithography process), or vapor etch,selective to all other materials (FIG. 10N). As illustrated in FIG. 10O,in the case of 712 being a conductive material connected to anelectrical circuit or an electrical source (e.g., a charge source), eachprobe tip formed by 712 on the stack (e.g., 781 and 782) can have acharge or an electrical field at the surface (e.g., 781 and 782), which(each probe tip) can be selected to have a positive charge or a negativecharge, or a positive electrical field or negative electrical field.Conversely, such probe tip can also sense various properties ofbiological subject being measured (e.g., electronic cloud, field,charge, or temperature when the probe tip is a thermal detector, orlight emission when the probe tip is an optical sensor). Usingelectrical circuit or electrical source, various combinations ofelectrical charge distribution or electrical field can be placed on themicro-device, as shown in FIG. 10O and FIG. 10P, which can be used tosort and trap various biological subjects such as a cell and a DNAmolecule. For instance, a biological subject with a charge distributioninverse of that in FIG. 10P can be trapped by the micro-device shown inFIG. 10P. An array of micro-devices with various charge distributions orelectrical field distributions can trap their respective biologicalsubjects in a high speed, which can serve as a sorting device. FIG. 10Qillustrates the use of a micro-device capable of trapping a DNA ormeasuring various properties (e.g., electrical, thermal, or opticalproperties) of a DNA, with each probe tip matched up spatially witheither a major groove or minor groove of a double helix DNA. FIG. 10Rillustrates how the probe tips are connected to electrical circuit,where only electrical wiring is shown. It should be noted that themicro-device shown in this example can be integrated onto a single chipwith one billion or more such micro-devices to trap and/or sort cells,DNAs, RNAs, proteins, and other biological subject in a high speed.

Another aspect of this invention relates to micro-indentation probes andmicro-probes for measuring a range of physical properties (such asmechanical properties) of biological subjects. Examples of themechanical properties include hardness, shear strength, elongationstrength, fracture stress, and other properties related to cell membranewhich is believed to be a critical component in disease diagnosis.

FIGS. 11A-11L illustrates a novel fabrication process flow formicro-devices capable of probing a range of properties of biologicalsubjects, such as mechanical properties of cell membrane (e.g.,mechanical strength of a cell membrane). In this process flow, amaterial 812 is first deposited onto a substrate 811, followed by thedeposition of another material 813 (FIG. 11A). Following patterning ofmaterial 813 using lithography and etch processes, a material 814 isdeposited (FIG. 11B) and planarized (FIG. 11C). Another layer ofmaterial 813 is next deposited and patterned using lithography and etchprocesses to remove portions of the material 813, followed by thedeposition and planarization of a material 815 (which can be apiezo-electrical material and can serve as a driver) (FIG. 11D). A layerof material 813 is next deposited, followed by deposition and patterningof yet another layer of 813, and deposition and planarization ofmaterial 816 (FIG. 11E). Next, material 816 is etched back to a reducedthickness, and patterned, followed by patterning of triple-layer ofmaterial 813 (FIG. 11F). Another layer of 814 is deposited (FIG. 11G)and planarized by chemical mechanical polishing (FIG. 11H), andpatterned (FIG. 11I). Finally, multiple layers of 813 are removed by wetetch, plasma etch, or vapor etch (FIG. 11J). FIG. 11K is a perspective,cross-sectional illustration of the micro-device in a planeperpendicular to that in FIG. 11J (90-degree rotation from FIG. 11J).FIG. 11L illustrates a micro-device with two micro-tips 871 and 872which can move in opposite directions when a voltage is applied topiezo-electrical drivers 881 and 882, which can be used to probebiological subjects such as cells.

FIGS. 12A-12B are illustrations of how micro-devices fabricated usingthe novel manufacturing process shown in FIGS. 11A-11L work. In FIGS.12A-12B, a micro-device 850 with two micro-probes 866 and 855 can movein opposite directions upon a force being applied (FIG. 12A). When thetips of the two probes are penetrated into a cell 870, as the distancebetween the two micro-probes is increased with the increasing appliedforce, the cell is stretched. Finally, as the applied force is reached acritical value, the cell is broken into two pieces (FIG. 12B). Thedynamic response of the cell to the applied force provides informationon the cell, particularly on the mechanical properties (e.g.,elasticity) of cell membrane. The force at the point in which the cellis torn apart reflects the strength of the cell and it may be called abreaking point: the greater the mechanical strength of the cell membraneis, the greater the force is at the breaking point.

Another novel approach provided by this invention is the use of phaselock-in measurement for disease detection, which reduces backgroundnoise and effectively enhances signal to noise ratio. Generally, in thismeasurement approach, a periodic signal is used to probe the biologicalsample and response coherent to the frequency of this periodic probesignal is detected and amplified, while other signals not coherent tothe frequency of the probe signal is filtered out, which therebyeffectively reduces background noise. In one of the embodiments in thisinvention, a probing micro-device can send a periodic probe signal(e.g., a pulsed laser team, a pulsed thermal wave, or an alternatingelectrical field) to a biological subject, response to the probe signalby the biological subject can be detected by a detecting micro-device.The phase lock-in technique can be used to filter out unwanted noise andenhance the response signal which is synchronized to the frequency ofthe probe signal. The following two examples illustrate the novelfeatures of time of flight detection arrangement in combination withphase lock-in detection technique to enhance weak signal and thereforedetection sensitivity in disease detection measurements.

FIGS. 13A-13B are illustrations of a novel time of flight detectionarrangement for disease detection applications. Specifically, FIG. 13Ashows a set-up for measuring biological subject 911 using detectionprobe 933 and clock generator 922, and FIG. 13B contains recorded signal921 due to structure 922, signal 931 recorded by signal probe 933, andprocessed signal 941 using a phase lock-in technique to filter out noisein recorded signal 931, where only response synchronized to clock signal921 is retained. In the setup shown in FIG. 13A, when a biologicalsubject such as a cell 911 passes a structure 922, it triggers a clearsignal (e.g., a light scattering signal if 922 is a light source, or asharp increase in voltage if 922 is an orifice structure in a resistor).Therefore, 922 can be used to register the arrival of the biologicalsubject, and as a clock when multiple structures of 922 are placed at aperiodic distance as shown in recorded signal trace 921 in FIG. 13B. Inaddition, when 922 is placed at a known distance in front of a probe933, it marks the arrival of a biological subject coming towards 933 andsignal response recorded at 933 is delayed by a time t from the signaltriggered by 922 where t equals distance between 922 and 933 divided bytraveling speed of the biological subject. As illustrated in FIG. 13B,signal 921 due to structure 922 is clear and periodic with periodicityproportional to distance between structure 922 s, while signal measuredby probe 933 has a high noise level and relatively weak signal relatedto the biological subject. With the utilization of phase lock-intechnique to filter out noise in recorded signal 931 by the detectionprobe 933 un-synchronized to clock signal 921, signal to noise ratio canbe greatly enhanced as shown in processed signal 941 in FIG. 13B.

FIGS. 14A-14B illustrate yet another time of flight disease detectionarrangement in which a clock signal generator 922, a probe signalgenerator 944, and a signal detection probe 955 are used, along withschematically recorded clock signal 921, total recorded response signal951 (except clock signal), and processed signal 952 using phase lock-intechnique. In this arrangement, a probe signal generator 944 is used toperturb the biological subject 911 (e.g., heating 911 up using anoptical beam, or adding an electrical charge to 911), and response tothe probe signal is subsequently measured as a function of time using anarray of detection probes 955. The filtered signal in 952 shows dynamicresponse to probe signal by 944 as it decays over time. Since normalcell and abnormal cell may respond differently to the probe signal, thisarrangement with proper micro-probes can be utilized to detect diseasessuch as cancer. In another embodiment utilizing this set-up (shown inFIGS. 14A-14B), the probe signal generator 944 can send a periodicsignal to the biological subject 911, detected response signal from thebiological subject by the detection probe 955 can be processed using thephase lock-in technique, with noise un-synchronized to the frequency ofthe probe signal filtered out and signal synchronized to the probesignal frequency amplified.

FIGS. 15A-15F are perspective illustrations of the novel multi-propertymicro-filter. A timed shutter 1502 is sandwiched between 2 pieces offilter membrane 1501 with wells. When a biological subject 1511 movesthrough the pathway of the well, it is first detected by the counter1512, which triggers the clock of the barrier panel 1502. Then thelarger cells will be filtered out, or blocked, by the filter's holes1001, while only the specific subjects with enough speed are able to getthrough the pathway 1503 before the timed shutter 1502 closes the filterpathway (see FIGS. 15C-15D). Otherwise it will be held back as the timedshutter 1502 moves to block the pathway as shown in FIGS. 15E-15F.

FIG. 16 illustrates a fluid delivery system that includes a pressuregenerator, a pressure regulator, a throttle valve, a pressure gauge, anddistributing kits. The pressure generator 1605 sustains fluid withdesired pressure, and the pressure is further regulated by the regulator1601 and then accurately manipulated by the throttle valve 1602.Meanwhile, the pressure is monitored at real time and fed back to thethrottle valve 1602 by the pressure gauge 1603. The regulated fluid isthen in parallel conducted into the multiple devices where a constantpressure is needed to drive the fluid sample.

FIGS. 17A-17C illustrate how a micro-device in a disease detectionapparatus of this invention can communicate, probe, detect, andoptionally treat and modify biological subjects at a microscopic level.FIG. 17A illustrates the sequence of cellular events from signalrecognition to cell fates determination. First, as the signals 1701 aredetected by receptors 1702 on the cell surface, the cell will integrateand encode the signals into a biologically comprehensible message, suchas calcium oscillation 1703. Consequently, corresponding proteins 1704in the cell will interact with the message, then be modified andtransform into ion-interacted proteins 1705 accordingly. Through thetranslocation, these modified proteins 1705 will pass the carriedmessage to the nuclear proteins, and the controlled modification onnuclear proteins will modulate the expression of gene 1707 whichincludes transcription, translation, epigenetic processes, and chromatinmodifications. Through messenger RNA 1709, the message is in turn passedto specific proteins 1710, thereby changing their concentration—whichthen determines or regulates a cell's decision or activities, such asdifferentiation, division, or even death.

FIG. 17B illustrates an apparatus of this invention which is capable ofdetecting, communicating with, treating, modifying, or probing a singlecell, by a contact or non-contact means. The apparatus is equipped withmicro-probes and micro-injectors which are addressed and modulated bythe controlling circuitry 1720. Each individual micro-injector issupplied with a separate micro-cartridge, which carries designedchemicals or compounds.

To illustrate how an apparatus of this invention can be used to simulatean intracellular signal, calcium oscillation is taken as an examplemechanism. First, a Ca²⁺-release-activated channel (CRAC) has to beopened to its maximal extent, which could be achieved by variousapproaches. In an example of the applicable approaches, a biochemicalmaterial (e.g., thapsigargin) stored in the cartridge 1724 is releasedby an injector 1725 to the cell, and the CRAC will open at the stimulusof the biological subject. In another example of the applicableapproaches, the injector 1724 forces a specific voltage on cellmembrane, which causes the CRAC to open as well.

The Ca²⁺ concentration of a solution in the injector 1728 can beregulated as it is a desirable combination of a Ca²⁺-containing solution1726, and a Ca²⁺ free solution 1727. While the injector 1730 contains aCa²⁺ free solution, then injectors 1728 and 1730 are alternatelyswitched on and off at a desired frequency. As such, the Ca²⁺oscillation is achieved and the content inside the cell membrane arethen exposed to a Ca²⁺ oscillation. Consequently, the cell's activitiesor fate is being manipulated by the regulated signal generated by theapparatus.

Meanwhile, the cell's response (e.g., in the form of an electrical,magnetic, electromagnetic, thermal, optical, acoustical, mechanicalproperty, or a combination thereof) can be monitored and recorded by theprobes integrated in this apparatus.

FIG. 17C illustrates another design of apparatus which is able to setupcommunication with a single cell. The apparatus is equipped withmicro-probes which are coated with biologically compatible compounds orelements, e.g., Ca, C, Cl, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na,S, or Zn. These probes can generate oscillating chemical signals withsuch an element or compound to interact with the cell, and results intoa response that affects the cell's activities or eventual fate asdescribe above. Likewise, this apparatus can probe and record the cell'sresponse (e.g., in the form of an electrical, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, electro-mechanical,electro-chemical, electro-chemical-mechanical, bio-chemical,bio-mechanical, bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, mechanical property, or acombination thereof) as well.

FIG. 18 illustrates the system block diagram of a disease detectionapparatus of this invention. This example includes a fluid deliveringsystem 1801, biological interface 1802, a probing and detecting device1803, a system controller 1805, a medical waste reclaiming and treatingsystem 1804. A biological sample or material is transported to theinterface 1802 by the fluid delivery system 1801, meanwhile the fluidparameters (or properties) are reported to the system controller 1805which comprises a logic processing unit, a memory unit, an applicationspecific chip, a sensor, a signal transmitter, and a signal receiver;and then the system controller 1805 can give further command to thesystem. The interface 1802 is an assembly which bridges a fluid sampleand the detecting device, and further monitors the parameters orproperties of the biological sample (e.g., pressure, temperature,stickiness, or flow rate) and then reports the date to the systemcontroller 1805 while distributing the biological sample to the probingand detecting device 1803 with a specified speed or pressure (which canbe commanded by the system controller 1805).

The system controller 1805 is the central commander and monitor of theentire system (or apparatus), where all the parameters and informationfrom various modules is processed and exchanged and the instructions aregiven out, and where the command is dispatched. The system controller1805 can include, e.g., a pre-amplifier, an electrical meter, a thermalmeter, a switching matrix, a system bus, a nonvolatile storage device, arandom access memory, a processor, and a user interface through whichthe user of the apparatus can manipulate, configure the apparatus, andread the operating parameters and final result. The pre-amplifier canprocess the raw signal to a recognizable signal for the meters. Themeters can force and measure corresponding signals which can be, e.g.,electrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical, or mechanical signals, orcombinations thereof. The switching matrix can switch the testingterminals of different arrays of the probe sub-apparatus. The userinterface includes input and output assemblies and is an assembly whichseals the fluid delivery system and the probing and detecting devicetogether.

The probing and detecting device 1803 is the core functional module ofthe disease detection apparatus of this invention as it is the unit thatprobes the biological sample and collects related cellular signals (orresponses). The waste reclaiming and treating system 1804 reclaims thewaste biological sample to protect the privacy of its biological host,and keeps it away from polluting the environment.

FIGS. 19B-19L illustrate a process flow for fabricating a micro-devicefor trapping, sorting, probing, measuring, treating, or modifying abiological subject (e.g., a single cell, a DNA or RNA molecule). A firstmaterial 1902 (e.g., a piezo-electrical conducting material) and asecond material 1903 (e.g., a conducting material) are sequentiallydeposited on a substrate 1901 (see FIGS. 19B and 19C). The secondmaterial 1903 is subsequently patterned by lithography and etchprocesses (see FIG. 19D). A third material 1904 is next deposited (asshown in FIG. 19E) and planarized (see FIG. 19F). A layer of a fourthmaterial 1905 is subsequently deposited (see FIG. 19G) and patterned asa hard mask (see FIG. 19H), followed by etch to remove the third andfirst materials from desired areas, which stops on the substrate 1901.FIG. 19I is a perspective illustration of the device, while FIG. 19J isa vertical illustration of the same device.

FIG. 19K illustrates the use of a micro-device capable of trapping a DNA1920 and measuring various properties (e.g., electrical, magnetic,physical, thermal, chemical, biological, bio-chemical, or opticalproperties) of a DNA. Each probe tip 1912 matches up spatially witheither a major groove or minor groove of a double helix DNA. Meanwhile,two probes (1911 and 1910) configured at the end of the trench can forceor measure signals to each strand end of the DNA's double helix. Theprobes can be made of a conducting material with optionally apiezo-electrical support structure, which can stretch forward andbackward at a desired distance. All the probes are numbered, addressed,and controlled by a controlling circuitry.

FIG. 19L shows a simplified form of the device illustrated in FIG. 19K.In this device, probe tips match spatially with interlaced grooves of adouble helix DNA. The number of groove intervals between the adjacentprobes is variable. If required, either DNA can be moved (for example,by pulling by probes 1910 and 1911) or the probes can move along thetrench direction, mapping out properties in a full or partial DNA.

FIGS. 20A-20C illustrate an apparatus of this invention that is capableof detecting or measuring the surface charge of a biological subject2010. It includes a channel, a pair of plates 2022, and a slit 2030which separates the channel into a top channel 2041 and a bottom channel2051. When a biological subject 2010 carrying a surface charge (positivecharge shown in FIG. 20A) passes through the channel, under theinfluence of the voltage applied on the plates 2022 (with positivevoltage at the top plate and negative at the bottom plate), it will movetowards the bottom plate as shown in FIG. 20B. Thus, the biologicalsubject 2010 will pass through the bottom channel 2051 when it reachesslit 2030. (If the biological subject 2010 carries a negative charge, itwould pass through the top channel 2041.) This way, a biological subjectwith unknown charge type (negative or positive) can be determined byusing this apparatus.

This device comprises at least 2 parts of channel, one of which ischannel 2060 where the biological subject is charged or modified, andthe other comprises at least one plate or slit to separate thebiological subjects (e.g., where the biological subjects are separated).

As surface charge will affect the shape of a biological subject, byusing novel and multiple plates, information on the shape and chargedistribution of biological subjects can be obtained. The generalprinciple and design of the micro-device can be extended to a broaderscope, thereby making it possible to obtain other information on thebiological subject via separation by applying other parameters such asion gradient, thermal gradient, optical beam, or another form of energy.

FIGS. 21A-21B illustrate another apparatus of this invention fordetecting or measuring microscopic properties of a biological subject2110 by utilizing a micro-device that includes a channel, a set ofprobes 2120, and a set of optical sensors 2132 (see, FIG. 21A). Thedetected signals by probes 2120 can be correlated to informationincluding images collected by the optical sensors 2132 to enhancedetection sensitivity and specificity. The optical sensors can be, e.g.,a CCD camera, a florescence light detector, a CMOS imaging sensor, orany combination.

Alternatively, a probe 2120 can be designed to trigger optical emissionsuch as florescence light emission 2143 in the targeted biologicalsubject such as diseased cells, which can then be detected by an opticalprobe 2132 as illustrated in FIG. 21B. Specifically, biological subjectscan be first treated with a tag solution which can selectively react todiseased cells. Subsequently, upon reacting (contact or non-contact)with probe 2120, optical emissions from diseased cells occur and can bedetected by optical sensors 2132. This novel process using themicro-devices of this invention is more sensitive than such conventionalmethods as traditional florescence spectroscopy as the emission triggerpoint is directly next to the optical probe and the triggered signal2143 can be recorded in real time and on-site, with minimum loss ofsignal.

FIG. 22 illustrates another embodiment of the apparatus of thisinvention, which can be used to separate biological subjects ofdifferent geometric size and detect their properties respectively. Itincludes at least an entrance channel 2210, a disturbing fluid channel2220, an accelerating chamber 2230, and two selecting channels 2240 and2250. The angle between 2220 and 2210 is between 0° and 180°. Thebiological subject 2201 flows in the x-direction from 2210 to 2230. Thebiocompatible distribution fluid 2202 flows from 2220 to 2230. Then thefluid 2202 will accelerate 2201 in y-direction. However, theacceleration correlates with the radius of the biological subjects andthe larger ones are less accelerated than the small ones. Thus, thelarger and smaller subjects are separated into different channels.Meanwhile, probes can be optionally assembled aside the sidewall of2210, 2220, 2230, 2240, and 2250. They could detect electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, physical, mechanical properties, or combinations thereof atthe microscopic level. In the mean time, if desired, a cleaning fluidcan also be injected into the system for dissolving and/or cleaningbiological residues and deposits (e.g., dried blood and protein) in thenarrow and small spaces in the apparatus, and ensuring smooth passage ofa biological subject to be tested through the apparatus.

The channel included in the apparatus of this invention can have a widthof, e.g., from 1 nm to 1 mm. The apparatus should have at least oneinlet channel and at least two outlet channels.

FIG. 23 shows another apparatus of this invention with an acousticdetector 2320 for measuring the acoustic property of a biologicalsubject 2301. This apparatus includes a channel 2310, and at least anultrasonic emitter and an ultrasonic receiver installed along thesidewall of the channel. When the biological subject 2301 passes throughthe channel 2310, the ultrasonic signal emitted from 2320 will bereceived after carrying information on 2301 by the receiver 2330. Thefrequency of the ultrasonic signal can be, e.g., from 2 MHz to 10 GHz,and the trench width of the channel can be, e.g., from 1 nm to 1 mm. Theacoustic transducer (i.e., the ultrasonic emitter) can be fabricatedusing a piezo-electrical material (e.g., quartz, berlinite, gallium,orthophosphate, GaPO₄, tourmalines, ceramics, barium, titanate, BatiO₃,lead zirconate, titanate PZT, zinc oxide, aluminum nitride, andpolyvinylidene fluorides).

FIG. 24 shows another apparatus of this invention that includes apressure detector for biological subject 2401. It includes at least onechannel 2410 and whereon at least one piezo-electrical detector 2420.When the biologic subject 2401 passes through the channel, thepiezo-electrical detector 2420 will detect the pressure of 2401,transform the information into an electrical signal, and send it out toa signal reader. Likewise, the trench width in the apparatus can be,e.g., from 1 nm to 1 mm, and the piezo-electrical material can be, e.g.,quartz, berlinite, gallium, orthophosphate, GaPO₄, tourmalines,ceramics, barium, titanate, BatiO₃, lead zirconate, titanate PZT, zincoxide, aluminum nitride, or polyvinylidene fluorides.

FIGS. 25A-25C show another apparatus of this invention that include aconcave groove 2530 between a probe couple, in the bottom or ceiling ofthe channel. When a biological subject 2510 passes through, the concave2530 can selectively trap the biological subject with particulargeometric characteristics and makes the probing more efficiently. Theshape of concave's projection can be rectangle, polygon, ellipse, orcircle. The probe could detect electrical, magnetic, electromagnetic,thermal, optical, acoustical, biological, chemical, physical, mechanicalproperties, or combinations thereof. Similarly, the trench width can be,e.g., from 1 nm to 1 mm. FIG. 25A is an up-down view of this apparatus,FIG. 25B is a side view, whereas FIG. 25C is a perspective view.

FIGS. 26A-26B are another apparatus of this invention that also includesconcave grooves 2630 (of a different shape from those shown in FIG. 25)on the bottom or ceiling of the channel. When a biological subject 2610passes through, the concave grooves 2630 will generate a turbulentfluidic flow, which can selectively trap the micro-biological subjectswith particular geometric characteristics. The probe could detect, e.g.,electrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, physical, mechanical properties, or a combinationthereof. The depth of the concave groove can be, e.g., from 10 nm to 1mm, and the channel width can be, e.g., from 1 nm to 1 mm.

FIG. 27 illustrates an apparatus of this invention with a steppedchannel 2710. When a biological subject 2701 passes through the channel2710, probe couples of different distances can be used to measuredifferent microscopic properties, or even the same microscopic atdifferent sensitivity at various steps (2720, 2730, 2740) with probeaside each step. This mechanism can be used in the phase lock-inapplication so that signal for the same microscopic property can beaccumulated. The probes can detect or measure microscopic electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, physical, mechanical properties, or combinations thereof.

FIG. 28 illustrates another apparatus of this invention with thermalmeters 2830. It includes a channel, a set of probes 2820, and a set ofthermal meters 2830. The thermal meters 2830 can be an infrared sensor,a transistor sub-threshold leakage current tester, or thermistor.

FIG. 29 illustrates a specific apparatus of this invention whichincludes carbon a nano-tube 2920 with a channel 2910 inside, probes 2940which can detect at the microscopic level an electrical, magnetic,electromagnetic, thermal, optical, acoustical, biological, chemical,physical, or mechanical property, or a combination thereof. The carbonnano-tube 2920 as shown contains a double-helix DNA molecule 2930. Thecarbon nano-tube can force and sense electrical signals by the probes2940 aside. The diameter of the carbon nano tube diameter can be, e.g.,from 0.5 nm to 50 nm, and its length can range from, e.g., 5 nm to 10mm.

FIGS. 30A-30G show an integrated apparatus of this invention thatincludes a detecting device (shown in FIG. 30A) and an optical sensor(shown in FIG. 30B) which can be, e.g., a CMOS image sensor (CIS), aCharge-Coupled Device (CCD), a florescence light detector, or anotherimage sensor. The detecting device comprises at least a probe and achannel, and the image device comprises at least 1 pixel. FIG. 30C andFIG. 30D illustrate the device with the detecting device and opticalsensor integrated. As illustrated in FIG. 30E, when biological subjects3001, 3002, 3003 pass through, the probe 3010 in the channel 3020, itselectrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, physical, mechanical property or a combinationthereof could be detected by the probe 3010 (see FIG. 30F), meanwhileits image could be synchronously recorded by the optical sensor andshown as different frames (or images) in FIG. 30G. Both the probedsignal and images are combined together to provide a diagnosis andenhanced detection sensitivity and specificity. Such a detecting deviceand an optical sensing device can be designed in a system-on-chip or bepackaged into one chip.

FIGS. 31A-D show an apparatus with a detecting micro-device (FIG. 31A)and a logic circuitry (FIG. 31B). The detecting device comprises atleast a probe and a channel, and the logic circuitry comprises anaddresser, an amplifier, and a RAM. When a biological subject 3101passes through the channel, its property could be detected by the probe3130, and the signal can be addressed, analyzed, stored, processed, andplotted in real time. FIG. 31C and FIG. 31D illustrate the device withdetecting device and Circuitry integrated. Similarly, the detectingdevice and the integrated circuit can be designed in a System-on-Chip orbe packaged into one chip.

FIGS. 32A-32C show an apparatus of this invention that comprises adetecting device (FIG. 32A) and a filter (FIG. 32B). When a biologicalsubject 3201 passes through the device, a filtration is performed in thefilter, and irrelevant objects can be removed. The remaining subjects'property can then be detected by the probe device (FIG. 31A). Thefiltration before probing will enhance the precision of the device. Thewidth of the channel can also range, e.g., from 1 nm to 1 mm.

FIG. 33 shows the geometric factors of DNA 3330 such as spacing in DNA'sminor groove (3310) have an impact on spatial distribution ofelectrostatic properties in the region, which in turn may impact localbiochemical or chemical reactions in the segment of this DNA. Byprobing, measuring, and modifying spatial properties of DNA (such as thespacing of minor groove) using the disclosed detector and probe 3320,one may detect properties such as defect of DNA, predictreaction/process at the segment of the DNA, and repair or manipulategeometric properties and therefore spatial distribution of electrostaticfield/charge, impacting biochemical or chemical reaction at the segmentof the DNA. For example, tip 3320 can be used to physically increasespacing of minor groove 3310.

FIG. 34 shows the fabrication process for a micro-device of thisinvention that has a flat cover atop of trench to form a channel. Thiswill eliminate the need for coupling two trenches to form a channel,which can be tedious for requiring perfect alignment. The cover can betransparent and allow observation with a microscope. It can comprise orbe made of silicon, SiGe, SiO₂, various types of glass, or Al₂O₃.

FIG. 35 is a diagram of an apparatus of this invention for detecting adisease in a biological subject. This apparatus includes apre-processing unit, a probing and detecting unit, a signal processing,and a disposal processing unit.

FIG. 36 shows an example of a sample filtration sub-unit in thepre-processing unit, which can separate the cells with differentdimensions or sizes. This device comprises at least one entrance channel3610, one disturbing fluid channel 3620, one accelerating chamber 3630,and two selecting channels (3640 and 3650). The angle 3660 between 3620and 3610 ranges from 0° to 180°.

The biological subject 3601 flows in the x direction from the entrancechannel 3610 to the accelerating chamber 3630. A bio-compatible fluid3602 flows from disturbing fluid channel 3620 to the acceleratingchamber 3630, it then accelerates the biological subject 3601 in they-direction. The acceleration correlates with the radius of thebiological subject and the larger ones are less accelerated than thesmaller ones. Then, the larger and smaller subjects are separated intodifferent selecting channels. Meanwhile, probes can be optionallyassembled on the sidewalls of the channels 3610, 3620, 3630, 3640, and3650. The probes could detect, at the microscopic level, electrical,magnetic, electromagnetic, thermal, optical, acoustical, biological,chemical, biochemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, physical, mechanical properties, orcombinations thereof.

FIGS. 37A-37B are diagrams of another example of a sample filtrationunit in the apparatus of this invention. 3701 represents small cells,while 3702 represents large cells. When a valve 3704 is open and anothervalve 3703 is closed, biological subjects (3701 and 3702) flow towardsexit A. Large cells that have larger size than the filtration hole areblocked against exit A, while small cells are flushed out through exitA. The entrance valve 3704 and exit A valve 3707 are then closed, and abio-compatible fluid is injected through the fluid entrance valve 3706.The fluid carries big cells are flushed out from exit B. The largercells are then analyzed and detected in the detection part of theinvention.

FIG. 38 is a diagram of a pre-processing unit of an apparatus of thisinvention. This unit includes a sample filtration unit, a rechargingunit or system for recharging nutrient or gas into the biologicalsubject, a constant pressure delivery unit, and a sample pre-probingdisturbing unit.

FIG. 39 is a diagram of an information or signal processing unit of anapparatus of this invention. This unit includes an amplifier (such as alock-in amplifier) for amplifying the signal, an A/D converter, and amicro-computer (e.g., a device containing a computer chip or informationprocessing sub-device), a manipulator, a display, and networkconnections.

FIG. 40 shows the integration of multiple signals which results incancellation of noise and enhancement of signal/noise ratio. In thisfigure, a biological 4001 is tested by Probe 1 during Δt between t1 andt2, and by Probe 2 during Δt between t3 and t4. 4002 is 4001's testedsignal from Probe 1, and 4003 is from Probe 2. Signal 4004 is theintegration result from signal 4002 and 4003. The noise cancels out eachother in certain extent and results in an improved signal strength orsignal/noise ratio. The same principle can be applied to data collectedfrom more than more than 2 micro-devices or probing units.

FIGS. 41A-41G show one embodiment of the fabrication processes flow ofthis invention for manufacturing a detection device with at least onedetection chamber and at least one detector. In this example, followingan optional process flow of fabricating data storage, data processingand analyzing components (including transistors, memory devices, logiccircuits, and RF devices), a material 4122 is first deposited onto asubstrate 4111, followed by the deposition of another material 4133(material for future detectors). Material 4133 can be selected fromelectrically conductive materials, piezo-electrical materials,semiconductor materials, thermal sensitive materials, ion emissionsensitive materials, pressure sensitive materials, mechanical stresssensitive materials, or optical materials. Optionally, it can alsoconsist of composite materials or a desired material stack. If required,an integrated detector with a set of sub-components can be placed atthis level. Material 4133 is next patterned using lithography and etchprocesses, forming a set of desired features shown in FIG. 41C. Anothermaterial 4144 is subsequently deposited, which can be the same as ordifferent from material 4122. Material 4122 can be an electricallyinsulating material such as oxide (SiO₂), doped oxide, silicon nitride,or polymer material. Next, the material 4144 is optionally planarizedusing polishing (e.g., using chemical mechanical polishing) or etch backprocess. The material stack is then patterned using lithography and etchprocesses, stopping on substrate 4111. Finally, as shown in FIG. 41G, acapping layer or the surface of another component 4155 is placed on topof the material stack (thereby sealing or capping it), forming anenclosed detection chamber 4166 with detector 4177 for biological sampledetection.

FIGS. 42A-42I illustrate another embodiment of the fabricating method ofthis invention for manufacturing a detection device with encloseddetection chambers, detectors, and channels for transporting biologicalsamples such as fluidic samples. In this embodiment, following anoptional process flow of fabricating data storage, data processing andanalyzing components (including transistors, memory devices, logiccircuits, and RF devices), a material 4222 is first deposited onto asubstrate 4211, followed by the deposition of another material 4233(material for future detectors). Material 4233 can be selected fromelectrical conductive materials, piezo-electrical materials,semiconductor materials, thermal sensitive materials, ion emissionsensitive materials, pressure sensitive materials, mechanical stresssensitive materials, or optical materials. Optionally, it can alsoinclude composite materials or a desired material stack. If required, anintegrated detector with a set of sub-components can be placed at thislevel.

Materials 4222 and 4233 are subsequently patterned using lithography andetch processes (FIG. 42C). These two layers (4222 and 4233) can bepatterned in separate patterning processes sequentially, or can bepatterned in the same process, pending on device design, types ofmaterials and etch chemistries. Substrate 4211 is next etched as shownin FIG. 42D, forming a recessed area (cavity) in 4211, in which stacks4222 and 4233 can be used as a hard mask during the etch process.

A material 4244 is deposited into the recessed area, and the portion ofthe material 4244 above the material 4233 is removed using a polishing(chemical or mechanical) or etch back process. Material 4244 can beselected from oxide, doped oxide, silicon nitride, and polymermaterials. A layer 4255 is then deposited onto material 4244 andpatterned to form small holes at selected locations. A wet or vapor etchis utilized next to remove material 4244, forming an enclosed detectionchamber 4266.

Optionally, as shown in FIG. 42I, the material 4222 is also removedusing wet or vapor etch process, forming channels 4288 connectingvarious detection chambers, thus forming detection chambers with adetector 4277 lined with the walls of the detection chamber and withgaseous or fluidic biological samples flowing through the chambers.Finally, the top surface of the detection chamber is sealed with anotherlayer of material (e.g., 4255).

FIGS. 43A-43C show a novel disease detection method of this invention inwhich at least one probe object is launched at a desired speed anddirection toward a biological subject, resulting in a collision. Theresponse(s) by the biological subject during and/or after the collisionis detected and recorded, which can provide detailed and microscopicinformation on the biological subject such as weight, density,elasticity, rigidity, structure, bonding (between different componentsin the biological subject), electrical properties such as electricalcharge, magnetic properties, structural information, and surfaceproperties. For example, for a same type of cell, it is expected that acancerous cell will experience a smaller traveling distance after thecollision than that of a normal cell due to its denser, greater weight,and possibly larger volume. As shown in FIG. 43A, a probe object 4311 islaunched towards a biological subject 4322. After the collision with theprobe object 4311, the biological subject 4322 may be pushed (scattered)out a distance depending on its properties as shown FIG. 43B.

FIG. 43C shows a schematic of a novel disease detection device with aprobe object launch chamber 4344, an array of detectors 4333, a probeobject 4322 and a biological subject to be tested 4311. In general, atest object can be an inorganic particle, an organic particle, acomposite particle, or a biological subject itself. The launch chambercomprises a piston to launch the object, a control system interfaced toan electronic circuit or a computer for instructions, and a channel todirect the object.

FIGS. 44A-44H illustrate a novel fabrication process for formingmultiple components with different materials at the same device level.First, a first material 4422 is deposited onto a substrate 4411 (seeFIG. 44A), followed by the deposition of a second material 4433. Thesecond material 4433 is next patterned to form at least a portion ofrecessed area in the layer 4433, using lithography and etch processes(see FIG. 44C). A third material 4444 is subsequently deposited. Thethird material can be the same as or different from the second material4422.

The third material directly above the second material is removed viaetch back and/or polishing (such as chemical mechanical polishing)processes (see FIG. 44E). Optionally, the third material is nextpatterned to form at least a portion of recessed area in layer 4444(FIG. 44F). A fourth material 4455 is then deposited. Optionally, theportion of the fourth material 4455 directly above the third material4444 or above both the second and third materials is removed via etchback and/or polishing (such as chemical mechanical polishing). The aboveprocess can keep repeating to form multiple features with the same ordifferent materials at the same device level. Hence, this process flowforms at least two components 4466 and 4477 with different materials orthe same materials at the same device level. For example, in oneembodiment, one component can be used as a prober and the other can beused as a detector.

FIG. 45 illustrates a method for detecting a disease in a biologicalsubject. A biological subject 4501 passes through the channel 4531 at aspeed v, and probe 4511 is a probe which can grossly detect theproperties of the biological subject at high speed.

Probe 4512 is a fine probing device which is coated by apiezo-electrical material. There is a distance ΔL between probe 4511 andprobe 4512.

When the biological subjects are tested when getting through 4511, ifthe entity is identified to be a suspected abnormal one, the systemwould trigger the piezo-electrical probe 4512 to stretch into thechannel and probe particular properties after a time delay of Δt. Andprobe 4512 retracts after the suspected entity passed through.

The probing device is capable of measuring at the microscopic level anelectrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject.

The width of the micro-channel can range from about 1 nm to about 1 mm.

FIGS. 46A-46B show a process of detecting a disease in a biologicalsubject. A biological subject 4601 passes through the channel 4631 at aspeed v. Probe 4611 is a probe which can grossly detect the propertiesof the biological subject at high speed. 4621 and 4622 arepiezo-electrical valves to control the micro-channel 4631 and 4632. 4612is a fine probing device which can probe biological properties moreparticularly. 4631 is flush channel to rush out normal biologicalsubjects. 4632 is detection channel where the suspected entities arefine detected in this channel.

When a biological subject is tested while getting through 4611, if it isnormal, the valve 4621 of the flush channel is open, while the detectionchannel valve 4622 is closed, the biological subject is flushed outwithout a time-consuming fine detection.

When the biological subject is tested while getting through 4611, if itis suspected to be abnormal or diseased, the valve 4621 of the flushchannel is closed, while the detection channel valve 4622 is open, thebiological subject is conducted to the detection channel for a moreparticular probing.

The width of the micro-channel can range from about 1 nm to about 1 mm.

The probing device is capable of measuring at the microscopic level anelectrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject.

FIGS. 47A-47B illustrate an arrayed biological detecting device. Asshown in FIG. 47A, 4701 are arrayed micro-channels which can get throughthe fluidics and biological subjects. 4702 are probing devices embeddedaside the channels. The sensors are wired by bit-lines 4721 andword-lines 4722. The signals are applied and collected by the decoderR\row-select 4742 and decoder column select 4741. As illustrated in FIG.47B, the micro-channel arrayed biological detecting device 4700 can beembedded in a macro-channel 4701. The micro-channel's dimension rangesfrom about 1 um to about 1 mm. The shape of the micro-channel can berectangle, ellipse, circle, or polygon.

The probing device is capable of measuring at the microscopic level anelectrical, magnetic, electromagnetic, thermal, optical, acoustical,biological, chemical, electro-mechanical, electro-chemical,electro-chemical-mechanical, bio-chemical, bio-mechanical,bio-electro-mechanical, bio-electro-chemical,bio-electro-chemical-mechanical, physical or mechanical property, or acombination thereof, of the biological subject.

FIGS. 48A-48B illustrate a device of the current invention for diseasedetection. 4801 is inlet of the detecting device, and 4802 is the outletof the device. 4820 is the channel where the biological subjects passthrough. 4811 is the optical component of the detecting device.

As illustrated in FIG. 48B, the optical component 4811 consists of anoptical emitter 4812 and an optical receiver 4813. The optical emitteremits an optical pulse (e.g. laser beam pulse), when the biologicalsubject 4801 passing through the optical component, and the opticalsensor detects the diffraction of the optical pulse, then identify themorphology of the entity.

FIGS. 49A-49I show a schedule for fabricating a piezo-electricalmicro-detector of this invention. Particularly, in FIG. 49A, a substrate4901 is deposited sequentially with a wet etching stop layer 4902 ofmaterial A, and with a sacrificial layer 4903 of material B. Thesacrificial layer 4903 is then patterned by the lithography and etchingprocesses. Shown in FIG. 49B, a layer 4904 of piezo-electrical materialC is then deposited onto the surface of the sacrificial layer 4903, andthen planarized. As shown in FIG. 49C, the layer 4904 is then patternedby lithography and etching processes. A second sacrificial layer 4905(which can be the same as or different from material B) and a second wetetching stop layer 4906 (which can be the same as or different frommaterial A) are subsequently deposited, as shown in FIG. 49D and FIG.49E. A patterning process using lithography and etching is performedthrough layers 4906 and 4905, and etching is stopped on thepiezo-electrical layer 4904. It is followed by depositing a conductivelayer 4907 of material D is deposited, and then patterning theconductive layer. See FIG. 49G. A patterning process is then followedand the etching stopped on the substrate, thereby forming a trench. SeeFIG. 49H. An isotropic wet etch selective to material B is thenfollowed, giving rise to a piezo-electrical probe (a cantilever) 4908.See FIG. 49I.

FIGS. 50A-50F show an example of the micro-device of this inventionpackaged and ready for integration with a sample delivery system anddata recording device. As illustrated in FIG. 50A, the device 5001 isfabricated by micro-electronics processes described herein and has atleast a micro-trench 5011, a probe 5022, and a bonding pad 5021. Thesurface of the device's top layer can include SixOyNz, Si, SixOy, SixNy,or a compound containing the elements of Si, O, and N. Component 5002 isa flat glass panel. In FIG. 50B, the flat panel 5002 is shown to bebonded with micro-device 5001 on the side of micro-trench. The bondingcan be achieved by a chemical, thermal, physical, optical, acoustical,or electrical means, or any combination thereof. FIG. 50C shows aconductive wire 5013 being bonded with the bonding pad from the side ofthe pads. As illustrated in FIG. 50D, the device 5001 is then packagedin a plastic cube with only conducting wires exposed. In FIG. 50E, aconical channel 5020 is carved through packaging material and connectingthe internal channel of the device. As illustrated in FIG. 50F, thelarger opening mouth of the conical channel makes it operational andconvenient to mount a sample delivery injector with the device, therebybetter enabling the delivery of sample from an injector with relativelylarge size of injector needle into device with relatively smallchannels.

FIGS. 51A-51F show another example of the micro-device of this inventionpackaged and ready for integration with a sample delivery system anddata recording device. As shown in FIG. 51A, a micro-device 5100 isfabricated by one or more micro-electronics processes as described inInternational Application No. PCT/US2011/042637, entitled “Apparatus forDisease Detection.” The micro-device 5100 has at least a micro-trench5104, a probe 5103, a connecting port 5102, and a bonding pad 5105. Onthe top of the micro-device 5100, the surface layer comprises SixOyNz,Si, SixOy, SixNy, or a compound consisting of Si, O, and N. The surfacelayer can be covered, and thus the micro-device 5100 is mounted, with aflat glass panel 5101. See FIG. 51B. The mounting can be by a chemical,thermal, physical, optical, acoustical, or electrical means. As shown inFIG. 51C, the conductive wire 5121 is bonded with bonding pad from theside of the pads. FIG. 51D illustrates that the micro-device 5100 canthen be packaged in a cube with only conducting wires exposed. Thepackaging cube can comprise a packaging material such as plastic,ceramic, metal, glass, or quartz. As shown in FIG. 51E, a tunnel 5141 isthen drilled into the cube until the tunnel reaches the connecting port5102. Further, as shown in FIG. 51F, the tunnel 5141 is then beingconnected to other pipes which can delivery a sample to be tested intothe micro-device 5100, and flush out the sample after the sample istested.

FIGS. 52A-52D show yet another example of the micro-device of thisinvention packaged and ready for integration with a sample deliverysystem and data recording device. As illustrated in FIG. 52A, device5200 is a micro-fluidic device which has at least one micro-channel5201. 5203 is a pipe that conducts a fluidic sample. The micro-channel5201 and the conducting pipe 5203 are aligned and submerged in a liquid,for example, water. FIG. 52B illustrates that, when the temperature ofthe liquid in which the micro-device and conducting pipe are submerged,is decreased to its freezing point or lower, the liquid solidifies intoa solid 5204. As illustrated in FIG. 52C, while the temperature of theliquid is maintained below the freezing point, the combination(including the solid 5204, the conducting pipe 5203, and the device5200) is enclosed into a packaging material 5205 whose meltingtemperature is higher than that of the solid 5204, with only theconducting pipe exposed. FIG. 52D shows that, after the temperature isincreased above the melting point of the solid 5204, the solid material5204 melts and becomes a liquid and is then exhausted from theconducting pipe 5203. The space 5206 wherein the solid material 5204once filled is now available or empty, and the channel 5201 and theconducting pipe 5203 are now connected through and sealed in the space5206.

FIGS. 53A-53C show a micro-device of this invention that has a channel(trench) and an array of micro sensors. In FIG. 53A, 5310 is a devicefabricated by microelectronics techniques; 5310 comprises micro-sensorarray 5301 and addressing and read-out circuitry 5302. The micro-sensorarray can include thermal sensors, piezo-electrical sensors,piezo-photronic sensors, piezo-optical electronic sensors, imagesensors, optical sensors, radiation sensors, mechanical sensors,magnetic sensors, bio-sensors, chemical sensors, bio-chemical sensors,acoustic sensors, or a combination of them. Examples of thermal sensorsinclude resistive temperature micro-sensors, micro-thermocouples,thermo-diodes and thermo-transistors, and SAW (surface acoustic wave)temperature sensor. Examples of image sensors include CCD (ChargeCoupled Device) and CIS (CMOS image sensor). Examples of radiationsensors include photoconductive devices, photovoltaic devices,pyro-electrical devices, and micro-antennas. Examples of mechanicalsensors include pressure micro-sensors, micro-accelerometers,micro-gyrometers, and micro flow-sensors. Examples of magnetic sensorsinclude magneto-galvanic micro-sensors, magneto-resistive sensors,magneto diodes and magneto-transistors. Examples of biochemical sensorscomprise conductimetric devices and potentiometric devices. FIG. 53Bshows a micro-device 5320 that includes a micro-trench 5321. Asillustrated in FIG. 53C, 5310 and 5320 are bonded together to form thenew micro-device 5330 which include a trench or channel 5331. Themicro-sensor array 5301 is exposed in the channel 5331.

FIGS. 54A-54E show another micro-device of this invention that comprisestwo panels one of which has an array of micro sensors and two microcylinders. Particularly, FIG. 54A shows a micro-device 5430 fabricatedby micro-electronic techniques, which comprises a micro-sensor array5431 and a read-out circuitry 5432, 5410 is another micro-sensor arraychip, and 5420 is a micro-cylinder. As illustrated in FIG. 54B, amicro-sensor array chip 5430 and two micro-cylinders 5420 are bonded toform a micro-trench with micro-sensor array exposed. In the micro-deviceillustrated in FIG. 54C, 5410 is flipped bonded onto the micro-trenchdevice 5431 and forms the device 5450. Device 5450 has a channel withmicro-sensor array embedded on top and bottom sides. FIG. 54Dillustrates the X-cross-section of the micro-device while FIG. 54Eillustrates the y-cross-section of the micro-.

FIGS. 55A-55C show a micro-device of this invention that comprises twopanels one of which has an array of micro sensors and two microcylinders both of which have a probing sensor. Particularly, in FIG.55A, device 5510 is fabricated by microelectronics techniques, whichcomprises a channel 5511, probe 5513 aside the channel, and a read-outcircuitry 5512. FIG. 55B illustrates the X-cross-section of the device,while FIG. 55C illustrates the y-cross-section of the device. Probe 5513can apply a disturbing signal to the entities passing through thechannel 5511.

FIGS. 56A-56B show another micro-device of this invention comprisingseveral “sub-devices.” Particularly, as illustrated in FIG. 56A, thedevice 5610 composes “sub-devices” 5611, 5612, 5613, and 5614, amongwhich 5611 and 5613 are devices which can apply disturbing signals, and5612 and 5614 are micro-sensor arrays. FIG. 56B illustrates thefunctioning diagram of the device 5610, when biological samples 5621under the test are passing through the channel 5610, they are disturbedby signal A applied by 5611, then being tested and recorded by detectingsensor array 1 of 5612. These biological samples are then disturbed bydisturb probe 5613 of array 2, and being tested by detecting sensor 5614of array 2. Disturbing probe 5611 of array 1 and disturbing probe 5613of array 2 can apply the same or different signals. Likewise, detectingsensor 5612 of array 1 and detecting sensor 5614 of array 2 can sense ordetect the same or different properties.

FIG. 57 shows an example of the micro-devices of this invention whichincludes an application specific integrated circuit (ASIC) chip with I/Opads. Specifically, as illustrated in FIG. 57, 5710 is a micro-devicewith a micro-fluidic channel 5712 and I/O pads 5711. 5720 is anApplication Specific Integrated Circuit (ASIC) chip with I/O pads 5721.5720 and 5710 can be wired together through the bonding of I/O pads. Assuch, with an ASIC circuitry 5720, the micro-fluidic detecting device5710 can perform more complicated computing and analytical functions.

FIGS. 58A-58G are diagrams of the underlying principal of themicro-device of this invention which functions by combining variouspre-screening and detection methods in unobvious ways. In FIG. 58A, abiological subject is first pre-screened for diseased biologicalentities, and then the diseased biological entities are separated fromthe normal (healthy or non-diseased) biological entities. The biologicalsubject containing the diseased biological entities separated from thenormal biological entities is detected using a desired disease detectionmethod. In FIG. 58B, a biological sample has gone through multiple,successive cell separation steps to concentrate diseased cells (orbiological entities). In FIG. 58C, after pre-screening to concentratediseased biological entities, bio-marker is used to detect diseasedbiological entities. In FIG. 58D, bio-marker is first used to separateout diseased biological entities and then the sorted out, diseasedbiological entities are further detected by various detection methods.In short, this process includes initial screening, initial separation,further screening, further separation, probing with one or moredisturbing signals or disturbing parameters (e.g., physical, mechanical,chemical, biological, bio-chemical, bio-physical, optical, thermal,acoustical, electrical, electro-mechanical, piezo-electrical,micro-electro-mechanical, or a combination thereof), and finallydetection. This sequence can repeat one or more times. The effect ofthis process is concentrating the diseased entities for improveddetection sensitivity and specificity, particularly for a biologicalsubject with a very low concentration of diseased entities, such ascirculating tumor cell (CTC).

In FIG. 58E through FIG. 58G, a set of novel processes include (a)pre-screening, pre-separation and initial separation for diseasedbiological entities, (b) further separation of diseased biologicalentities, (c) optionally carry out initial detection, and (d) detectionusing various processes and detection methods. In the pre-separationprocess, one of the embodiments utilizes nano-particles or nano-magneticparticles attached with bio-markers to sort out diseased biologicalentities. During pre-separation process, the diseased biologicalentities are concentrated for higher concentration, which will makefurther separation and/or following detection easier. The biologicalsample following pre-separation process can go through furtherseparation process to further enhance the concentration of diseasedbiological entities. Finally, the biological sample gone through thepre-separation and follow-up separation steps will go through detectionstep(s), in which various detection techniques and processes can be usedto determine diseased biological entities and their types. In someembodiments, multiple detection steps can be utilized to detect diseasedbiological entities.

FIG. 59A shows a cross-sectional view of a channel (5911) into which abiological subject can flow. FIG. 59B shows an outside view of thechannel, along which an array of detectors (5922) are installed alongthe path of the flow of the biological subject. Alternatively, bothprobes and detectors can be installed to both disturb the biologicalsubject to be detected and detect response signals from such disturbsignals. FIG. 59C shows a cross-section of the wall of the channel,where detectors (5922) are mounted through to contact the biologicalsubject to be detected and also are making contact with the outsideworld (e.g., to connect to a detection circuitry).

FIG. 60A shows a biological subject (6033) to be detected passingthrough a channel (6011) aligned with detectors (6022) along itspassage. The detectors can be the same type of detectors, or acombination of various detectors. Further, probes capable of sending outprobing or disturbing signals to the biological subject to be detectedcan also be implemented along the channels, along with detectors whichcan detect response from the biological subject which has been probed ordisturbed by the probe. The detected signals can be acoustical,electrical, optical (e.g., imaging), biological, bio-chemical,bio-physical, mechanical, bio-mechanical, electro-magnetic,electro-mechanical, electro-chemical-mechanical,electro-chemical-physical, thermal, and thermal-mechanical propertyrelated signals, or a combination of them. FIG. 60B shows an example ofa set of detected signals (e.g., images, pressures, or electricalvoltages) (6044) along the path of the biological subject, whichrecorded its behavior and properties as it passes through the channel.For example, for an optical detector, the size of the circle shown inthe FIG. 60B could mean the optical emission from the biological subject(such as an optical emission from a florescence component attached tothe biological subject), the strength of a strain (pressure) acting onthe side wall of the channel detected by a piezo-electric detector or apiezo-photronic detector, or thermal emission from the biologicalsubject detected by a thermal detector or an IR sensor. Such detectedsignals can be solely from the biological subject as it passes throughthe channel, or responses from the biological subject to a disturbing orprobing signal by the probe.

Like FIG. 60B, FIG. 60C through FIG. 60E show additional examples ofvarious detected signal patterns (6044) as the biological subject passesthrough the channel and is detected by the novel detectors and processesdisclosed in the application.

FIGS. 61A-61N illustrate a device fabrication process flow andassociated device structures. In this process, a first material (6122)is deposited onto a substrate 6111 (see FIG. 61A), followed by theformation of an etch mask 6133 which could be a photoresist, a hardmask, or another type of mask (see FIG. 61C). The first material is nextpatterned, with un-masked area of the first material removed (see FIG.61D). Examples of suitable methods for removing the first materialinclude dry etch and wet etch. Following the removal of the maskmaterial (see FIG. 61E), a second material (6144) is deposited (see FIG.61F). A portion of the second material is next removed, with the secondmaterial above the first material substantially removed and the secondmaterial in the recessed area of the first material remaining (see FIG.61G). Examples of suitable methods for removing the second materialinclude etch-back using dry etch and wet etch, and chemical mechanicalpolishing. A third material 6155 is subsequently deposited (see FIG.61H), and a small opening is patterned (FIG. 61I), optionally utilizinglithography and etch processes, or optical ablation processes. Followingcreating the opening in the third material, the second material issubstantially removed (FIG. 61I), utilizing methods including but notlimited to wet etching, vapor etching, optical processing, and hightemperature heating (to evaporate the second material). With theseprocesses, various structures can be formed in the devices, whichinclude but are not limited to channels, probes, detectors, chambers,cavities, and other types of novel and traditional structures andfeatures. FIGS. 61I and 61J show a cross-sectional view and a top viewof a device with micro-channels 6166 and a chamber 6177, respectively.

To enhance the biological subject processing (such as treating,pre-separation, separation, sorting, probing and detecting) capabilityand throughput, more features and higher number of channels, chambers,probes, detectors, and channels can be fabricated on the same devicethrough building multiple layers of the above disclosed devicestructures, thereby increasing number of biological entities to beprocessed and detected. Specifically, the process flow described abovecan be repeated to build multiple layers. FIG. 61K shows a three-layerdevice with three layers of channels for carrying a biological subjectand chambers for various applications such as for pre-separating,separating, probing, and detection biological entities.

Instead of building a large number of layers on the same substrate (forexample, over 20 layers), it is sometimes advantageous to build amoderate number of layers and then stack multiple chips each withmultiple layers on it into a device with many layers on it (usingtechnologies such as flip chip and other packaging processes). FIG. 61Lshows two chips (6188 and 6199) with three layers in each chip. In somecases, backside of the chip needs to be thinned before stacking themtogether. After stacking multiple chips (such as the two chips 6188 and6199 in FIG. 61L) where each chip has multiple layers fabricated usingthe novel design and processes disclosed in this application, anintegrated device with a large number of layers comprising variousstructures and features (such as chambers or channels for pre-sorting,pre-screening, pre-separation, sorting, screening, separation, probing,and detection), as shown in FIGS. 61M and 61N.

To effectively sorting, separating, screening, probing, or detecting ofdiseased biological entities, a chamber (or chambers) integrated withvarious channels can be deployed as shown FIG. 62A, where incomingsample flowing into a chamber (6211) first. In the chamber, varioustechniques such as bio-markers and nano-technology (magnetic beads ornano-particles with bio-markers attached to them) based processes can beused to sort out, screen, and separate out the diseased biologicalentities. For example, a biological sample flowing from the left intothe chamber can have its diseased entities separated out in the chamber,and passed downward through the bottom channel, while its normalentities can continue to flow from the chamber in the right handdirection, through the channel in the right side of the chamber.Depending upon the design, the diseased entities, having entered intothe chamber on the left, can also be separated out in the chamber, andcontinue on towards right and flow into the channel on the right side ofthe chamber, while normal entities will continue to flow down toward andthrough the channel at the bottom of the chamber. FIG. 62B showsmultiple chambers integrated with channels in which biological entitiescan be sorted, screened, separated, probed or detected. In theapplication of screening and separation, the multiple chambers can carryout multiple screening and separation steps. As shown in FIG. 62B, for abiological sample flowing from the left toward the right direction, itwill enter into the first chamber on the left (6233) and under go afirst screening and separation. The biological sample can continue toflow towards the right, enter into the second chamber, the chamber onthe right (6244), and undergo a second screening and further separation.In this way, through a multi-staged screening and separation process,the concentration of a diseased entity can be successively enhancedwhich can be helpful for a sensitive final or late stage detection. Thistype of device design and process could be very useful for defection ofa biological sample with an initially very low concentration of diseasedentity population, such as for the detection of circulating tumor cell(CTC) which is typically in the concentration of one part in one billioncells or 10 billion cells.

To significantly speed up the sorting, screening, probing and detectionoperations using the disclosed device and process, a high number ofdesired structures such as those discussed in FIG. 63 can be fabricatedsimultaneously on the same chip as shown in FIG. 63.

FIG. 64 shows another novel device layout for sorting, screening,separating, probing and detecting diseased biological entities, in whicha desired component or multiple components through the middle channelinto the middle chamber 6411 can play a wide range of roles. Forexample, the component flowing into the middle chamber could be abio-marker which can be freshly added into the top chamber 6422 andbottom chamber 6433 when its (bio-marker) concentration needs to beadjusted. The timing, flow rate, and amount of component in the middlechamber 6411 need to be added into the top and bottom chambers (6422 and6433) can be pre-programmed or controlled via a computer or software inreal time. The component into the middle chamber 6412 could also benano-particles or magnetic beads attached to bio-markers. In anothernovel embodiment, the component into the middle chamber 6411 could be adisturbing agent which will disturb the biological subject or samples tobe detected in the top and bottom chambers.

Tests were carried out in the laboratory with the micro-devices of thisinvention on certain cancerous tissue samples (with multiple samples foreach type of cancer) although the micro-devices of this invention can beused for detection of other types of cancer or other types of treatment.In the tests, healthy control samples were obtained from animals with noknown cancer disease at the time of collection and no history ofmalignant disease. Both cancerous samples and healthy control sampleswere collected and cultured in the same type of culture solution. Thecultured samples were then mixed with a dilution buffer and diluted tothe same concentration. The diluted samples were maintained at the roomtemperature for different time intervals and processed within a maximumof 6 hours after being recovered. The diluted samples were tested at theroom temperature (20˜23° C.) and in the humidity of 30%˜40%. The sampleswere tested with a micro-device of this invention under the sameconditions and stimulated by the same pulse signal.

The tests show that, in general, the control groups' tested (measured)values (i.e., measured values in relative units for the testingparameter) were lower than the cancerous or diseased groups. Under thesame stimulation (in terms of stimulation type and level) with astimulating or probing signal applied by a probing unit of the testedmicro-devices of this invention, the difference shown in the measuredvalues between the control groups and the cancerous groups became muchmore significant, e.g., ranging from 1.5 times to almost 8 times interms of level of increase in such difference, compared with thatwithout simulation. In other words, the cancerous groups' response tothe stimulating signal was much higher than that of the control groups.Thus, the micro-devices of this invention have been proven to be able tosignificantly enhance the relative sensitivity and specificity in thedetection and measurement of diseased cells, in comparison to thecontrol or healthy cells.

Further, the test results show that in terms of the novel parameterutilized by the micro-device of this invention, the cancerous group andthe control group showed significantly different response. Suchdifference is significantly greater than the measurement noise. Therewas a large window to separate the control groups from the cancerousgroups, showing a high degree of sensitivity of the novel measurementmethod and apparatus.

While for the purposes of demonstration and illustration, the abovecited novel, detailed examples show how microelectronics and/ornano-fabrication techniques and associated process flows can be utilizedto fabricate highly sensitive, multi-functional, powerful, andminiaturized detection devices, the principle and general approaches ofemploying microelectronics and nano-fabrication technologies in thedesign and fabrication of high performance detection devices have beencontemplated and taught, which can and should be expanded to variouscombination of fabrication processes including but not limited to thinfilm deposition, patterning (lithography and etch), planarization(including chemical mechanical polishing), ion implantation, diffusion,cleaning, various materials, combination of processes and steps, andvarious process sequences and flows. For example, in alternativedetection device design and fabrication process flows, the number ofmaterials involved can be fewer than or exceed four materials (whichhave been utilized in the above example), and the number of processsteps can be fewer or more than those demonstrated process sequences,depending on specific needs and performance targets. For example, insome disease detection applications, a fifth material such as abiomaterial-based thin film can be used to coat a metal detection tip toenhance contact between the detection tip and a biological subject beingmeasured, thereby improving measurement sensitivity.

Applications for the detection apparatus and methods of this inventioninclude detection of diseases (e.g., in their early stage), particularlyfor serious diseases like cancer. Since cancer cell and normal celldiffer in a number of ways including differences in possible microscopicproperties such as electrical potential, surface charge, density,adhesion, and pH, novel micro-devices disclosed herein are capable ofdetecting these differences and therefore applicable for enhancedcapability to detect diseases (e.g., for cancer), particularly in theirearly stage. In addition micro-devices for measuring electricalpotential and electrical charge parameters, micro-devices capable ofcarrying out mechanical property measurements (e.g., density) can alsobe fabricated and used as disclosed herein. In mechanical propertymeasurement for early stage disease detection, the focus will be on themechanical properties that likely differentiate disease or cancerouscells from normal cell. As an example, one can differentiate cancerouscells from normal cells by using a detection apparatus of this inventionthat is integrated with micro-devices capable of carrying outmicro-indentation measurements.

Although specific embodiments of this invention have been illustratedherein, it will be appreciated by those skilled in the art that anymodifications and variations can be made without departing from thespirit of the invention. The examples and illustrations above are notintended to limit the scope of this invention. Any combination ofdetection apparatus, micro-devices, fabrication processes, andapplications of this invention, along with any obvious their extensionor analogs, are within the scope of this invention. Further, it isintended that this invention encompass any arrangement, which iscalculated to achieve that same purpose, and all such variations andmodifications as fall within the scope of the appended claims.

All publications or patent applications referred to above areincorporated herein by reference in their entireties. All the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings) may be replaced by alternative features servingthe same, equivalent or similar purpose, unless expressly statedotherwise. Thus, unless expressly stated otherwise, each featuredisclosed is one example of a generic series of equivalent or similarfeatures.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. All publications referenced herein are incorporated byreference in their entireties.

What is claimed is:
 1. A method for detecting a disease at a very lowconcentration of diseased biological subject, comprising contacting thediseased biological subject with a micro-device which comprises: a firstsorting unit capable of directly detecting an intrinsic property of thebiological subject at the microscopic level and sorting the biologicalsubject by the detected intrinsic property; optionally, a channel fortransporting a suspect of diseased biological subject to detection unitfor further examination; optionally, a channel for transporting anon-suspect of diseased biological subject for disposal or for othertypes of usage; a first detection unit capable of detecting the same ordifferent property of the sorted biological subject at the microscopiclevel; optionally, a channel for transporting a suspect of diseasedbiological subject from detection unit back to sorting unit or detectionunit for further sorting or detection; optionally, a channel fortransporting a non-suspect of diseased biological subject from detectionunit for disposal or for other types of usage; and a first layer ofmaterial having an exterior surface and an interior surface, wherein theinterior surface defines a first channel in which the biological subjectflows through the first sorting unit, and then the sorted biologicalsubject flows from the first sorting unit to the first detection unit;wherein the first sorting unit and the first detection unit areintegrated into the first layer of material and positioned to be atleast partially exposed in the channel.
 2. The method of claim 1,wherein the diseased biological subject is cells, a sample of an organor tissue, DNA, RNA, virus, or protein.
 3. The method of claim 2,wherein the cells are circulating tumor cells or cancer cells.
 4. Themethod of claim 1, wherein the biological subject is a sample of blood,urine, sweat, tear, or saliva.
 5. The method of claim 1, wherein thebiological subject is contained in a media and transported into thefirst channel of micro-device.
 6. The method of claim 1, wherein themicro-device further comprises a probing unit which is capable ofapplying a probing signal to the biological subject or a media in whichthe biological subject is contained, thereby changing the nature orvalue of a property of the biological subject or of the media.
 7. Themethod of claim 6, wherein the micro-device further comprising apre-screening unit which is capable of pre-screening a diseasedbiological subject from a non-diseased biological subject based on thedifference in a property between a diseased biological subject and anon-diseased biological subject.
 8. The method of claim 1, wherein thedisease is cancer.
 9. The method of claim 8, wherein the cancer isbreast cancer, lung cancer, esophageal cancer, intestine cancer, cancerrelated to blood, liver cancer, stomach cancer, or circulating tumorcells.